target dna fragments taq dna polymerase  (New England Biolabs)


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    New England Biolabs target dna fragments taq dna polymerase
    Alkaline Phosphatase Calf Intest CIP
    Alkaline Phosphatase Calf Intest CIP 5 000 units
    https://www.bioz.com/result/target dna fragments taq dna polymerase/product/New England Biolabs
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    Images

    1) Product Images from "Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V"

    Article Title: Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-81

    Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.
    Figure Legend Snippet: Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Produced, Plasmid Preparation

    2) Product Images from "A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases"

    Article Title: A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-7-r69

    Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
    Figure Legend Snippet: Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).

    Techniques Used: Injection, Polymerase Chain Reaction

    3) Product Images from "A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases"

    Article Title: A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-7-r69

    Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
    Figure Legend Snippet: Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).

    Techniques Used: Injection, Polymerase Chain Reaction

    4) Product Images from "Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes"

    Article Title: Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

    Journal: The Plant Journal

    doi: 10.1111/tpj.14450

    Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .
    Figure Legend Snippet: Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .

    Techniques Used: SPR Assay, Flow Cytometry, BAC Assay, Transformation Assay, Polymerase Chain Reaction, Amplification, Marker, Plasmid Preparation, Modification, Purification, Selection, Clone Assay, Sequencing, Construct, Conjugation Assay

    5) Product Images from "Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V"

    Article Title: Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-81

    Colony PCR screening to detect successful cloning of Mitf. Presence of the Mitf coding region inside the plasmid vector pBluescript II KS(+) in correct orientation detected by colony PCR and analytical gel electrophoresis. From 40 individually tested colonies, 39 were judged positive as evident from the amplification of a DNA fragment (expected size: 1621 bp). Colonies from the same E. coli strain transformed with plasmid DNA lacking the Mitf coding region served as negative control (NC).
    Figure Legend Snippet: Colony PCR screening to detect successful cloning of Mitf. Presence of the Mitf coding region inside the plasmid vector pBluescript II KS(+) in correct orientation detected by colony PCR and analytical gel electrophoresis. From 40 individually tested colonies, 39 were judged positive as evident from the amplification of a DNA fragment (expected size: 1621 bp). Colonies from the same E. coli strain transformed with plasmid DNA lacking the Mitf coding region served as negative control (NC).

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Amplification, Transformation Assay, Negative Control

    Scheme for the generation of cohesive ends. In addition to regions complementary to the insert DNA sequence (1) , oligonucleotides are designed with overhangs comprising the 4 bp cohesive part of a restriction site combined with deoxyinosine (dI) at the third position from the 5′ end. Primer annealing and extension during a PCR leads to amplification of the desired target fragment (2) , which carries the dI residues (bold, orange) in its termini. The pairing properties of the universal base will generate a sequence distribution at the corresponding site of the opposing strand (indicated as 'N’). Purified PCR products are treated with EndoV, which cleaves the second phosphodiester bond 3′ to dI (3) . The target DNA fragment (4) is obtained by spin column-based or agarose gel purification, respectively, removing the weakly bound residues of ssDNA. Carrying cohesive ends with 5′ phosphates, the insert fragment is now suitable for ligation to vector DNA fragments created by conventional restriction enzyme treatment (in the depicted case SacI and KpnI).
    Figure Legend Snippet: Scheme for the generation of cohesive ends. In addition to regions complementary to the insert DNA sequence (1) , oligonucleotides are designed with overhangs comprising the 4 bp cohesive part of a restriction site combined with deoxyinosine (dI) at the third position from the 5′ end. Primer annealing and extension during a PCR leads to amplification of the desired target fragment (2) , which carries the dI residues (bold, orange) in its termini. The pairing properties of the universal base will generate a sequence distribution at the corresponding site of the opposing strand (indicated as 'N’). Purified PCR products are treated with EndoV, which cleaves the second phosphodiester bond 3′ to dI (3) . The target DNA fragment (4) is obtained by spin column-based or agarose gel purification, respectively, removing the weakly bound residues of ssDNA. Carrying cohesive ends with 5′ phosphates, the insert fragment is now suitable for ligation to vector DNA fragments created by conventional restriction enzyme treatment (in the depicted case SacI and KpnI).

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Ligation, Plasmid Preparation

    Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.
    Figure Legend Snippet: Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Produced, Plasmid Preparation

    Insert DNA fragment generation using a proofreading DNA polymerase. PfuTurbo C x Hotstart polymerase was used for PCR amplification of insert DNA fragments using two deoxyinosine-containing oligonucleotides. Analytical agarose gel electrophoresis was performed with PCR products comprising the ampicillin resistance cassette ( 1114 bp, A) , the mRFP1 reporter device ( 830 bp, B) , and the Mitf coding sequence ( 1270 bp, C) . Annealing temperatures which were used for PCR cycling are indicated relative to T m for each lane.
    Figure Legend Snippet: Insert DNA fragment generation using a proofreading DNA polymerase. PfuTurbo C x Hotstart polymerase was used for PCR amplification of insert DNA fragments using two deoxyinosine-containing oligonucleotides. Analytical agarose gel electrophoresis was performed with PCR products comprising the ampicillin resistance cassette ( 1114 bp, A) , the mRFP1 reporter device ( 830 bp, B) , and the Mitf coding sequence ( 1270 bp, C) . Annealing temperatures which were used for PCR cycling are indicated relative to T m for each lane.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Sequencing

    Cohesive dsDNA ends created in this study. In order to ligate insert DNA fragments efficiently with a linearized target plasmid vector, both molecules have to carry compatible cohesive ends. For the vector DNA, 5′ recessed ends are created by conventional restriction enzyme treatment. Names and recognition sequences of the enzymes used in this study are listed. For other enzymes, please refer to REBASE [ 60 ]. Endonucleolytic cleavage positions are depicted as vertical dashes. Insert DNA fragments with compatible cohesive ends are created by PCR and subsequent endonuclease V treatment (as illustrated in Figure 2 ). The 5′ ends of the PCR primers and the termini of the corresponding PCR products differ from the shown sequences: They lack the first nucleotide (shown in red) and carry deoxyinosine instead of the residue shown in orange. EndoV treatment of the PCR product results in 5′ recessed ends shown in bold letters with yellow background. If the residue highlighted in grey is omitted from the oligonucleotide design, ligation of the insert fragments with linearized plasmid DNA does not reconstitute the restriction enzyme site.
    Figure Legend Snippet: Cohesive dsDNA ends created in this study. In order to ligate insert DNA fragments efficiently with a linearized target plasmid vector, both molecules have to carry compatible cohesive ends. For the vector DNA, 5′ recessed ends are created by conventional restriction enzyme treatment. Names and recognition sequences of the enzymes used in this study are listed. For other enzymes, please refer to REBASE [ 60 ]. Endonucleolytic cleavage positions are depicted as vertical dashes. Insert DNA fragments with compatible cohesive ends are created by PCR and subsequent endonuclease V treatment (as illustrated in Figure 2 ). The 5′ ends of the PCR primers and the termini of the corresponding PCR products differ from the shown sequences: They lack the first nucleotide (shown in red) and carry deoxyinosine instead of the residue shown in orange. EndoV treatment of the PCR product results in 5′ recessed ends shown in bold letters with yellow background. If the residue highlighted in grey is omitted from the oligonucleotide design, ligation of the insert fragments with linearized plasmid DNA does not reconstitute the restriction enzyme site.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Ligation

    6) Product Images from "A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases"

    Article Title: A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-7-r69

    Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
    Figure Legend Snippet: Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).

    Techniques Used: Injection, Polymerase Chain Reaction

    7) Product Images from "A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases"

    Article Title: A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-7-r69

    Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
    Figure Legend Snippet: Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).

    Techniques Used: Injection, Polymerase Chain Reaction

    8) Product Images from "A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases"

    Article Title: A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-7-r69

    Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
    Figure Legend Snippet: Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs . (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type s qt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).

    Techniques Used: Injection, Polymerase Chain Reaction

    Heritable deletions in the sqt locus that result in RNA-null alleles . (a) PCR on single wild-type or sqt deletion mutant embryos (using primers indicated in Figure 2a) shows a 220 bp fragment in a s qt sg32 locus-deletion embryo, and a 380 bp fragment in TSS deleted sqt sg27 mutant embryo. Sometimes a larger approximately 500 bp fragment is observed in sqt sg27 /+ heterozygous embryos, but the sequence is identical to the 478 bp product. (b) Percentage of embryos with sqt mutant phenotypes in sqt cz35/+ , sqt sg27/+ , sqt sg32/+ and sqt sg7/+ in-crosses and mating of sqt cz35/+ with sqt sg27/+ , sqt sg32/+ and sqt sg7/+ . The cz35 allele is an approximately 1.9 kb insertion in sqt exon 1; the sg27 allele is a 98 bp deletion of sqt TSS sequences; sg32 allele is a whole locus deletion of sqt ; the sg7 ZFN allele harbors a GGCC insertion in sqt exon 2. (c-j) DIC images of 24 h wild-type (c), sqt cz35/cz35 (d), sqt sg27/cz35 (e), sqt sg32/cz35 (f), sqt sg7/cz35 (g), sqt sg27/sg27 (h), sqt sg32/sg32 (i), and sqt sg7/sg7 (j) embryos; scale bar in (c), 100 μm. (k) UCSC genome browser view of the sqt locus and neighboring genomic region. (l,m) RT-PCR with primers to detect expression of sqt RNA and transcripts of neighboring genes, eif4ebp1 , rnf180 , and htr1ab , shows lack of sqt RNA expression in sqt sg27/sg27 (l) and sqt sg32/sg32 (m) embryos whereas all neighboring gene transcripts are expressed at wild-type levels. Actin ( act ) expression was used as control. In contrast, both un-spliced and spliced sqt RNA is detected in wild-type and heterozygous embryos.
    Figure Legend Snippet: Heritable deletions in the sqt locus that result in RNA-null alleles . (a) PCR on single wild-type or sqt deletion mutant embryos (using primers indicated in Figure 2a) shows a 220 bp fragment in a s qt sg32 locus-deletion embryo, and a 380 bp fragment in TSS deleted sqt sg27 mutant embryo. Sometimes a larger approximately 500 bp fragment is observed in sqt sg27 /+ heterozygous embryos, but the sequence is identical to the 478 bp product. (b) Percentage of embryos with sqt mutant phenotypes in sqt cz35/+ , sqt sg27/+ , sqt sg32/+ and sqt sg7/+ in-crosses and mating of sqt cz35/+ with sqt sg27/+ , sqt sg32/+ and sqt sg7/+ . The cz35 allele is an approximately 1.9 kb insertion in sqt exon 1; the sg27 allele is a 98 bp deletion of sqt TSS sequences; sg32 allele is a whole locus deletion of sqt ; the sg7 ZFN allele harbors a GGCC insertion in sqt exon 2. (c-j) DIC images of 24 h wild-type (c), sqt cz35/cz35 (d), sqt sg27/cz35 (e), sqt sg32/cz35 (f), sqt sg7/cz35 (g), sqt sg27/sg27 (h), sqt sg32/sg32 (i), and sqt sg7/sg7 (j) embryos; scale bar in (c), 100 μm. (k) UCSC genome browser view of the sqt locus and neighboring genomic region. (l,m) RT-PCR with primers to detect expression of sqt RNA and transcripts of neighboring genes, eif4ebp1 , rnf180 , and htr1ab , shows lack of sqt RNA expression in sqt sg27/sg27 (l) and sqt sg32/sg32 (m) embryos whereas all neighboring gene transcripts are expressed at wild-type levels. Actin ( act ) expression was used as control. In contrast, both un-spliced and spliced sqt RNA is detected in wild-type and heterozygous embryos.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Activated Clotting Time Assay

    9) Product Images from "Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes"

    Article Title: Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

    Journal: The Plant Journal

    doi: 10.1111/tpj.14450

    Recombineering with ccdB gene cassettes. The work flow of recombineering with the ccdB exchange cassettes is illustrated in the example of replacement of the stop codon of CDKD;3 (At1g18040) gene by the GFP coding sequences (Figure S2 e). The CDKD;3 BAC clone (T10F20) carrying a CmR marker is introduced into the recombineering host E. coli SW102 and the presence of the target gene is verified by PCR amplification with gene‐specific primers (green arrows) flanking its stop codon. In the first step of recombineering (1), the SW102 (BAC T10F20) strain is transformed with the DNA fragment of KmR‐araC‐ccdB cassette (2.7 kb), which is PCR amplified with primers carrying 50 nt flanks of the target stop codon (blue and red bars). KmR transformants are selected and regrown in LB‐Km−0.5% glucose medium without selecting for the BAC CmR marker, to enhance the loss of BACs lacking the ccdB insertion. Colonies carrying only BACs with the ccdB insertion are identified by PCR (2.7 kb + space between the gene‐specific primers). In the second step (2), the obtained SW102 (BAC: ccdB ) strain is transformed with a DNA fragment of GFP coding region, which is PCR amplified with primers carrying the 50 nt flanks of the stop codon (0.82 kb). Transformants are selected and enriched for the BAC CmR marker in LB medium containing 0.2% arabinose to induce the suicide ccdB gene expression. Exchange of the ccdB marker with the GFP cassette is monitored by colony PCR (0.72 kb + space between the gene‐specific primers). In the third step (3), the modified plant gene is moved by gap‐repair into an Agrobacterium binary vector. When using pGAPKm or pGAPHyg (Bitrián et al ., 2011 ; Figure S2 a), two BAC segments flanking the modified gene (usually located upstream and downstream of neighbouring genes) are PCR amplified as Eco RI‐ Sal I and Sal I‐ Bam HI fragments and inserted into Eco RI− Bam HI sites of pGAPs. Subsequently, the vectors are linearized by Sal I, phosphatase treated and transformed into SW102 (BAC:GFP). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH5α or DH10B. The presence of modified plant gene is verified by restriction enzyme fingerprinting and sequencing with the gene‐specific primers. The verified clone is transformed to the E. coli donor stain MFDpir ΔTIV lacIq and the conjugated into Agrobacterium GV3101 (pMP90RK) for plant transformation. To save time, the gap‐repair step (3) is performed with PCR‐amplifiable pGAPBRKm and pGAPBRHyg vectors as shown in Figure 2 b. BACs carrying a KmR marker are similarly modified using either the SpR‐ccdB or CmR‐ccdB cassette. The latter was used for modification of CDKF;1 , CDKD;1 and CDKD;2 genes (Figure S2 b–d). The ccdB exchange cassettes can be similarly inserted into any position of a target gene and replaced with DNA fragments carrying point mutations, codon exchanges or deletions.
    Figure Legend Snippet: Recombineering with ccdB gene cassettes. The work flow of recombineering with the ccdB exchange cassettes is illustrated in the example of replacement of the stop codon of CDKD;3 (At1g18040) gene by the GFP coding sequences (Figure S2 e). The CDKD;3 BAC clone (T10F20) carrying a CmR marker is introduced into the recombineering host E. coli SW102 and the presence of the target gene is verified by PCR amplification with gene‐specific primers (green arrows) flanking its stop codon. In the first step of recombineering (1), the SW102 (BAC T10F20) strain is transformed with the DNA fragment of KmR‐araC‐ccdB cassette (2.7 kb), which is PCR amplified with primers carrying 50 nt flanks of the target stop codon (blue and red bars). KmR transformants are selected and regrown in LB‐Km−0.5% glucose medium without selecting for the BAC CmR marker, to enhance the loss of BACs lacking the ccdB insertion. Colonies carrying only BACs with the ccdB insertion are identified by PCR (2.7 kb + space between the gene‐specific primers). In the second step (2), the obtained SW102 (BAC: ccdB ) strain is transformed with a DNA fragment of GFP coding region, which is PCR amplified with primers carrying the 50 nt flanks of the stop codon (0.82 kb). Transformants are selected and enriched for the BAC CmR marker in LB medium containing 0.2% arabinose to induce the suicide ccdB gene expression. Exchange of the ccdB marker with the GFP cassette is monitored by colony PCR (0.72 kb + space between the gene‐specific primers). In the third step (3), the modified plant gene is moved by gap‐repair into an Agrobacterium binary vector. When using pGAPKm or pGAPHyg (Bitrián et al ., 2011 ; Figure S2 a), two BAC segments flanking the modified gene (usually located upstream and downstream of neighbouring genes) are PCR amplified as Eco RI‐ Sal I and Sal I‐ Bam HI fragments and inserted into Eco RI− Bam HI sites of pGAPs. Subsequently, the vectors are linearized by Sal I, phosphatase treated and transformed into SW102 (BAC:GFP). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH5α or DH10B. The presence of modified plant gene is verified by restriction enzyme fingerprinting and sequencing with the gene‐specific primers. The verified clone is transformed to the E. coli donor stain MFDpir ΔTIV lacIq and the conjugated into Agrobacterium GV3101 (pMP90RK) for plant transformation. To save time, the gap‐repair step (3) is performed with PCR‐amplifiable pGAPBRKm and pGAPBRHyg vectors as shown in Figure 2 b. BACs carrying a KmR marker are similarly modified using either the SpR‐ccdB or CmR‐ccdB cassette. The latter was used for modification of CDKF;1 , CDKD;1 and CDKD;2 genes (Figure S2 b–d). The ccdB exchange cassettes can be similarly inserted into any position of a target gene and replaced with DNA fragments carrying point mutations, codon exchanges or deletions.

    Techniques Used: Flow Cytometry, BAC Assay, Marker, Polymerase Chain Reaction, Amplification, Transformation Assay, Expressing, Modification, Plasmid Preparation, Selection, Sequencing, Staining, SPR Assay

    Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .
    Figure Legend Snippet: Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .

    Techniques Used: SPR Assay, Flow Cytometry, BAC Assay, Transformation Assay, Polymerase Chain Reaction, Amplification, Marker, Plasmid Preparation, Modification, Purification, Selection, Clone Assay, Sequencing, Construct, Conjugation Assay

    10) Product Images from "Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes"

    Article Title: Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

    Journal: The Plant Journal

    doi: 10.1111/tpj.14450

    Recombineering with  ccdB  gene cassettes. The work flow of recombineering with the  ccdB  exchange cassettes is illustrated in the example of replacement of the stop codon of  CDKD;3  (At1g18040) gene by the GFP coding sequences (Figure   S2 e). The  CDKD;3  BAC clone (T10F20) carrying a CmR marker is introduced into the recombineering host  E. coli  SW102 and the presence of the target gene is verified by PCR amplification with gene‐specific primers (green arrows) flanking its stop codon. In the first step of recombineering (1), the SW102 (BAC T10F20) strain is transformed with the DNA fragment of KmR‐araC‐ccdB cassette (2.7 kb), which is PCR amplified with primers carrying 50 nt flanks of the target stop codon (blue and red bars). KmR transformants are selected and regrown in LB‐Km−0.5% glucose medium without selecting for the BAC CmR marker, to enhance the loss of BACs lacking the  ccdB  insertion. Colonies carrying only BACs with the  ccdB  insertion are identified by PCR (2.7 kb + space between the gene‐specific primers). In the second step (2), the obtained SW102 (BAC: ccdB ) strain is transformed with a DNA fragment of GFP coding region, which is PCR amplified with primers carrying the 50 nt flanks of the stop codon (0.82 kb). Transformants are selected and enriched for the BAC CmR marker in LB medium containing 0.2% arabinose to induce the suicide  ccdB  gene expression. Exchange of the  ccdB  marker with the GFP cassette is monitored by colony PCR (0.72 kb + space between the gene‐specific primers). In the third step (3), the modified plant gene is moved by gap‐repair into an  Agrobacterium  binary vector. When using pGAPKm or pGAPHyg (Bitrián  et al .,   2011 ; Figure   S2 a), two BAC segments flanking the modified gene (usually located upstream and downstream of neighbouring genes) are PCR amplified as  Eco RI‐ Sal I and  Sal I‐ Bam HI fragments and inserted into  Eco RI− Bam HI sites of pGAPs. Subsequently, the vectors are linearized by  Sal I, phosphatase treated and transformed into SW102 (BAC:GFP). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into  E. coli  DH5α or DH10B. The presence of modified plant gene is verified by restriction enzyme fingerprinting and sequencing with the gene‐specific primers. The verified clone is transformed to the  E. coli  donor stain MFDpir ΔTIV lacIq and the conjugated into  Agrobacterium  GV3101 (pMP90RK) for plant transformation. To save time, the gap‐repair step (3) is performed with PCR‐amplifiable pGAPBRKm and pGAPBRHyg vectors as shown in Figure   2 b. BACs carrying a KmR marker are similarly modified using either the SpR‐ccdB or CmR‐ccdB cassette. The latter was used for modification of  CDKF;1 ,  CDKD;1  and  CDKD;2  genes (Figure   S2 b–d). The  ccdB  exchange cassettes can be similarly inserted into any position of a target gene and replaced with DNA fragments carrying point mutations, codon exchanges or deletions.
    Figure Legend Snippet: Recombineering with ccdB gene cassettes. The work flow of recombineering with the ccdB exchange cassettes is illustrated in the example of replacement of the stop codon of CDKD;3 (At1g18040) gene by the GFP coding sequences (Figure  S2 e). The CDKD;3 BAC clone (T10F20) carrying a CmR marker is introduced into the recombineering host E. coli SW102 and the presence of the target gene is verified by PCR amplification with gene‐specific primers (green arrows) flanking its stop codon. In the first step of recombineering (1), the SW102 (BAC T10F20) strain is transformed with the DNA fragment of KmR‐araC‐ccdB cassette (2.7 kb), which is PCR amplified with primers carrying 50 nt flanks of the target stop codon (blue and red bars). KmR transformants are selected and regrown in LB‐Km−0.5% glucose medium without selecting for the BAC CmR marker, to enhance the loss of BACs lacking the ccdB insertion. Colonies carrying only BACs with the ccdB insertion are identified by PCR (2.7 kb + space between the gene‐specific primers). In the second step (2), the obtained SW102 (BAC: ccdB ) strain is transformed with a DNA fragment of GFP coding region, which is PCR amplified with primers carrying the 50 nt flanks of the stop codon (0.82 kb). Transformants are selected and enriched for the BAC CmR marker in LB medium containing 0.2% arabinose to induce the suicide ccdB gene expression. Exchange of the ccdB marker with the GFP cassette is monitored by colony PCR (0.72 kb + space between the gene‐specific primers). In the third step (3), the modified plant gene is moved by gap‐repair into an Agrobacterium binary vector. When using pGAPKm or pGAPHyg (Bitrián et al ., 2011 ; Figure  S2 a), two BAC segments flanking the modified gene (usually located upstream and downstream of neighbouring genes) are PCR amplified as Eco RI‐ Sal I and Sal I‐ Bam HI fragments and inserted into Eco RI− Bam HI sites of pGAPs. Subsequently, the vectors are linearized by Sal I, phosphatase treated and transformed into SW102 (BAC:GFP). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH5α or DH10B. The presence of modified plant gene is verified by restriction enzyme fingerprinting and sequencing with the gene‐specific primers. The verified clone is transformed to the E. coli donor stain MFDpir ΔTIV lacIq and the conjugated into Agrobacterium GV3101 (pMP90RK) for plant transformation. To save time, the gap‐repair step (3) is performed with PCR‐amplifiable pGAPBRKm and pGAPBRHyg vectors as shown in Figure  2 b. BACs carrying a KmR marker are similarly modified using either the SpR‐ccdB or CmR‐ccdB cassette. The latter was used for modification of CDKF;1 , CDKD;1 and CDKD;2 genes (Figure  S2 b–d). The ccdB exchange cassettes can be similarly inserted into any position of a target gene and replaced with DNA fragments carrying point mutations, codon exchanges or deletions.

    Techniques Used: Flow Cytometry, BAC Assay, Marker, Polymerase Chain Reaction, Amplification, Transformation Assay, Expressing, Modification, Plasmid Preparation, Selection, Sequencing, Staining, SPR Assay

    Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .
    Figure Legend Snippet: Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .

    Techniques Used: SPR Assay, Flow Cytometry, BAC Assay, Transformation Assay, Polymerase Chain Reaction, Amplification, Marker, Plasmid Preparation, Modification, Purification, Selection, Clone Assay, Sequencing, Construct, Conjugation Assay

    11) Product Images from "Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes"

    Article Title: Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

    Journal: The Plant Journal

    doi: 10.1111/tpj.14450

    Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .
    Figure Legend Snippet: Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .

    Techniques Used: SPR Assay, Flow Cytometry, BAC Assay, Transformation Assay, Polymerase Chain Reaction, Amplification, Marker, Plasmid Preparation, Modification, Purification, Selection, Clone Assay, Sequencing, Construct, Conjugation Assay

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    Article Snippet: .. Lysates of Vero cells that had been infected with wild-type HSV-2 186 at an MOI of 3 for 24 h and lysates of HEK293T cells that had been transfected with pEGFP-EF-1δ(F) were treated with calf intestinal alkaline phosphatase (CIP) (New England BioLabs) as described previously ( ). .. Vero and U2OS cells were infected with each of the recombinant viruses at an MOI of 0.0001, and plaque sizes were determined as described previously ( ).

    Amplification:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Ligation:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Infection:

    Article Title: Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis
    Article Snippet: .. Lysates of Vero cells that had been infected with wild-type HSV-2 186 at an MOI of 3 for 24 h and lysates of HEK293T cells that had been transfected with pEGFP-EF-1δ(F) were treated with calf intestinal alkaline phosphatase (CIP) (New England BioLabs) as described previously ( ). .. Vero and U2OS cells were infected with each of the recombinant viruses at an MOI of 0.0001, and plaque sizes were determined as described previously ( ).

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Purification:

    Article Title: “Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest
    Article Snippet: .. One microgram of bait RNA was therefore dephosphorylated using 1 U CIP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs, Evry, France) at 37 °C for 60 min and RNAs were purified by phenol-chloroform extraction. .. RNA Sample Preparation RNA samples that will be hybridized to the bait were isolated from MCF-7 breast cancer cells or primary myoblasts with TRI reagent (Sigma) as previously described [ , ].

    Concentration Assay:

    Article Title: HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase *
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    Incubation:

    Article Title: HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase *
    Article Snippet: .. Lysates (30 μg) were incubated with or without calf intestinal alkaline phosphatase at the concentration of 1 unit/μg of protein in CutSmart buffer, both obtained from New England Biolabs (Ipswich, MA). .. The reaction was supplemented with 1 m m protease inhibitor mixture and PMSF and incubated for 30 min at 37 °C.

    Plasmid Preparation:

    Article Title: Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge
    Article Snippet: .. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, the plasmid Miniprep kit, and the DNA fragment gel extraction kit were purchased from New England Biolabs and used according to the manufacturer's specifications. .. B. abortus strain 2308 and the vaccine strain RB51 were obtained from the National Veterinary Services Laboratory, USDA (Ames, IA).

    De-Phosphorylation Assay:

    Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty
    Article Snippet: .. Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions. ..

    Article Title: Biochemical and Genetic Requirements for Function of the Immune Response Regulator BOTRYTIS-INDUCED KINASE1 in Plant Growth, Ethylene Signaling, and PAMP-Triggered Immunity in Arabidopsis [C] [C] [W]
    Article Snippet: .. Protein dephosphorylation was performed using calf intestinal alkaline phosphatase (CIP) according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units of CIP (as indicated)/μg protein. .. Total RNA was isolated with Trizol reagent according to the manufacturer’s instructions (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Gel Extraction:

    Article Title: Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge
    Article Snippet: .. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, the plasmid Miniprep kit, and the DNA fragment gel extraction kit were purchased from New England Biolabs and used according to the manufacturer's specifications. .. B. abortus strain 2308 and the vaccine strain RB51 were obtained from the National Veterinary Services Laboratory, USDA (Ames, IA).

    Derivative Assay:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

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    New England Biolabs target dna fragments taq dna polymerase
    Robust PCR-amplification of insert <t>DNA</t> fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by <t>Taq</t> polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.
    Target Dna Fragments Taq Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.

    Journal: BMC Biotechnology

    Article Title: Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

    doi: 10.1186/1472-6750-13-81

    Figure Lengend Snippet: Robust PCR-amplification of insert DNA fragments using deoxyinosine-containing primers. Analytical agarose gel electrophoresis of PCR products produced by Taq polymerase using either plasmid DNA (A) or E. coli colonies (B) as template material. Relative to the calculated T m , annealing temperatures used for PCR cycling are indicated for each lane.

    Article Snippet: PCR-based amplification of target DNA fragments Taq DNA polymerase and dNTP mix were obtained from New England Biolabs (Frankfurt am Main, Germany).

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Produced, Plasmid Preparation