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  • 91
    Jena Bioscience restriction enzymes bglii
    Restriction Enzymes Bglii, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction enzymes bglii
    Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between <t>BglII</t> and <t>NcoI.</t> P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.
    Restriction Enzymes Bglii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher restriction enzymes bglii
    A schematic representation of a double homologues recombination event between transformation vector (pCCATCH) and chloroplast genome ( a ). The sequence of the cat CH gene, under control of the C. merolae chloroplast promoter P <t>dna</t> K, (marked as white, curved rectangle ) of the dna K gene, was integrated into the chloroplast molecule at the selected position between the rpl 32 - psb A genes. The solid black rectangles represent the algal chloroplast sequence, also present in the transformation vector, flanking the cat CH cassettes and functioning as facilitators of double homologues recombination. b Verification of the insertion of the cat CH cassette into the rpl 32- psb A region of the chloroplast molecule via PCR and subsequent agarose gel electrophoresis of the PCR products. The PCR reaction was performed on the total DNA, isolated from the wild type ( WT ) and stable chloroplast transformant lines (transformed by B biolistic bombardment or P PEG method). Two sets of primers were used: 5UTF-5UTR amplifying a 4612 bp long product (marked as x) and 3UTF-3UTR amplifying a 3655 bp long product (marked as y). These two PCR products encompass the regions between cat CH gene and fragments from beyond the upstream or downstream homologous region in the transformation vector. The presence of the cat CH was confirmed by a PCR reaction with catCHStuI-catCHKpnI primers pair amplifying a 672 bp long product (marked as z). To exclude the possibility of unwarranted, random integration of the vector or its continuous presence in the cell, a fragment of the plasmid ori region was amplified with the pABB20R-pABB20L primer pair, yielding a 3420 bp long product, only in case of the vector control, thereby confirming the absence of any residual plasmid (marked as v). M molecular marker. c Southern blot hybridization of PCR products generated by the 5UTF-5UTR and 3UTF-3UTR primer pairs with the CATCH probe (being the PCR product generated with catCHStuI-catCHKpnI primers pair). The total DNA, isolated from the stable chloroplast transformants with the integrated pCCATCH, obtained via biolistic bombardment ( B ) or PEG method ( P ) was used as a templates for PCR. The correct integration of the transformation vector was confirmed by the size of apparent bands (4612 bp and 3655 bp, see the upper part ) and additionally by Southern blot hybridization with cat probe (the lower part ). d The Southern blot hybridization of <t>BglII-digested</t> total DNA with the psb A probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with BglII restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 8 µg of DNA. The DNA was transferred onto a nitrocellulose membrane and hybridized with psbA probe. The transformant-derived psbA containing band was calculated to have the length of 4.2 kbp. The WT control fragment was calculated to form a 2.5 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared psb A probe. The semiquantitative results showed two bands (pointed out with arrows ) in the P and B lines, representing the wild type ptDNA band and 1.8 kbp larger (containing P dnaK cat CH cassette) recombined ptDNA band. The presence of both types of plastids demonstrated heterogeneity of the obtained transformant lineages. e The Southern blot hybridization of EcoRI-digested total DNA with the cat CH probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with EcoRI restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 5 µg of DNA. The cat containing band was calculated to have the length of 10 kbp. Additionally, a positive control was added by digesting the transformation plasmid with EcoRI and AatII. This fragment was calculated to range ~10 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared 32 P enriched probe. The developed film shows no signal in the WT wells and one dominant signal in the transformed cells lines, confirming that the transformation vector had successfully recombined in a unique and single locus of the plastid without any unwarranted, illegitimate recombination with the genome or the plastid. The positive control is overexposed as the molar contribution of the cat sequence in the transformation vector is great deal higher than in the plastid
    Restriction Enzymes Bglii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs restriction enzyme bgli
    A schematic representation of a double homologues recombination event between transformation vector (pCCATCH) and chloroplast genome ( a ). The sequence of the cat CH gene, under control of the C. merolae chloroplast promoter P <t>dna</t> K, (marked as white, curved rectangle ) of the dna K gene, was integrated into the chloroplast molecule at the selected position between the rpl 32 - psb A genes. The solid black rectangles represent the algal chloroplast sequence, also present in the transformation vector, flanking the cat CH cassettes and functioning as facilitators of double homologues recombination. b Verification of the insertion of the cat CH cassette into the rpl 32- psb A region of the chloroplast molecule via PCR and subsequent agarose gel electrophoresis of the PCR products. The PCR reaction was performed on the total DNA, isolated from the wild type ( WT ) and stable chloroplast transformant lines (transformed by B biolistic bombardment or P PEG method). Two sets of primers were used: 5UTF-5UTR amplifying a 4612 bp long product (marked as x) and 3UTF-3UTR amplifying a 3655 bp long product (marked as y). These two PCR products encompass the regions between cat CH gene and fragments from beyond the upstream or downstream homologous region in the transformation vector. The presence of the cat CH was confirmed by a PCR reaction with catCHStuI-catCHKpnI primers pair amplifying a 672 bp long product (marked as z). To exclude the possibility of unwarranted, random integration of the vector or its continuous presence in the cell, a fragment of the plasmid ori region was amplified with the pABB20R-pABB20L primer pair, yielding a 3420 bp long product, only in case of the vector control, thereby confirming the absence of any residual plasmid (marked as v). M molecular marker. c Southern blot hybridization of PCR products generated by the 5UTF-5UTR and 3UTF-3UTR primer pairs with the CATCH probe (being the PCR product generated with catCHStuI-catCHKpnI primers pair). The total DNA, isolated from the stable chloroplast transformants with the integrated pCCATCH, obtained via biolistic bombardment ( B ) or PEG method ( P ) was used as a templates for PCR. The correct integration of the transformation vector was confirmed by the size of apparent bands (4612 bp and 3655 bp, see the upper part ) and additionally by Southern blot hybridization with cat probe (the lower part ). d The Southern blot hybridization of <t>BglII-digested</t> total DNA with the psb A probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with BglII restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 8 µg of DNA. The DNA was transferred onto a nitrocellulose membrane and hybridized with psbA probe. The transformant-derived psbA containing band was calculated to have the length of 4.2 kbp. The WT control fragment was calculated to form a 2.5 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared psb A probe. The semiquantitative results showed two bands (pointed out with arrows ) in the P and B lines, representing the wild type ptDNA band and 1.8 kbp larger (containing P dnaK cat CH cassette) recombined ptDNA band. The presence of both types of plastids demonstrated heterogeneity of the obtained transformant lineages. e The Southern blot hybridization of EcoRI-digested total DNA with the cat CH probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with EcoRI restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 5 µg of DNA. The cat containing band was calculated to have the length of 10 kbp. Additionally, a positive control was added by digesting the transformation plasmid with EcoRI and AatII. This fragment was calculated to range ~10 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared 32 P enriched probe. The developed film shows no signal in the WT wells and one dominant signal in the transformed cells lines, confirming that the transformation vector had successfully recombined in a unique and single locus of the plastid without any unwarranted, illegitimate recombination with the genome or the plastid. The positive control is overexposed as the molar contribution of the cat sequence in the transformation vector is great deal higher than in the plastid
    Restriction Enzyme Bgli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bgli restriction enzyme
    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific <t>BglI</t> restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. <t>PCR</t> products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.
    Bgli Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega bglii restriction enzymes
    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific <t>BglI</t> restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. <t>PCR</t> products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.
    Bglii Restriction Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa bglii restriction enzymes
    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific <t>BglI</t> restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. <t>PCR</t> products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.
    Bglii Restriction Enzymes, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest bglii
    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific <t>BglI</t> restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. <t>PCR</t> products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.
    Fastdigest Bglii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xhoi bglii restriction enzymes
    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific <t>BglI</t> restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. <t>PCR</t> products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.
    Xhoi Bglii Restriction Enzymes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.

    Journal: Applied and Environmental Microbiology

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    doi: 10.1128/AEM.00227-15

    Figure Lengend Snippet: Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.

    Article Snippet: The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Techniques: Expressing, BAC Assay

    A schematic representation of a double homologues recombination event between transformation vector (pCCATCH) and chloroplast genome ( a ). The sequence of the cat CH gene, under control of the C. merolae chloroplast promoter P dna K, (marked as white, curved rectangle ) of the dna K gene, was integrated into the chloroplast molecule at the selected position between the rpl 32 - psb A genes. The solid black rectangles represent the algal chloroplast sequence, also present in the transformation vector, flanking the cat CH cassettes and functioning as facilitators of double homologues recombination. b Verification of the insertion of the cat CH cassette into the rpl 32- psb A region of the chloroplast molecule via PCR and subsequent agarose gel electrophoresis of the PCR products. The PCR reaction was performed on the total DNA, isolated from the wild type ( WT ) and stable chloroplast transformant lines (transformed by B biolistic bombardment or P PEG method). Two sets of primers were used: 5UTF-5UTR amplifying a 4612 bp long product (marked as x) and 3UTF-3UTR amplifying a 3655 bp long product (marked as y). These two PCR products encompass the regions between cat CH gene and fragments from beyond the upstream or downstream homologous region in the transformation vector. The presence of the cat CH was confirmed by a PCR reaction with catCHStuI-catCHKpnI primers pair amplifying a 672 bp long product (marked as z). To exclude the possibility of unwarranted, random integration of the vector or its continuous presence in the cell, a fragment of the plasmid ori region was amplified with the pABB20R-pABB20L primer pair, yielding a 3420 bp long product, only in case of the vector control, thereby confirming the absence of any residual plasmid (marked as v). M molecular marker. c Southern blot hybridization of PCR products generated by the 5UTF-5UTR and 3UTF-3UTR primer pairs with the CATCH probe (being the PCR product generated with catCHStuI-catCHKpnI primers pair). The total DNA, isolated from the stable chloroplast transformants with the integrated pCCATCH, obtained via biolistic bombardment ( B ) or PEG method ( P ) was used as a templates for PCR. The correct integration of the transformation vector was confirmed by the size of apparent bands (4612 bp and 3655 bp, see the upper part ) and additionally by Southern blot hybridization with cat probe (the lower part ). d The Southern blot hybridization of BglII-digested total DNA with the psb A probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with BglII restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 8 µg of DNA. The DNA was transferred onto a nitrocellulose membrane and hybridized with psbA probe. The transformant-derived psbA containing band was calculated to have the length of 4.2 kbp. The WT control fragment was calculated to form a 2.5 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared psb A probe. The semiquantitative results showed two bands (pointed out with arrows ) in the P and B lines, representing the wild type ptDNA band and 1.8 kbp larger (containing P dnaK cat CH cassette) recombined ptDNA band. The presence of both types of plastids demonstrated heterogeneity of the obtained transformant lineages. e The Southern blot hybridization of EcoRI-digested total DNA with the cat CH probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with EcoRI restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 5 µg of DNA. The cat containing band was calculated to have the length of 10 kbp. Additionally, a positive control was added by digesting the transformation plasmid with EcoRI and AatII. This fragment was calculated to range ~10 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared 32 P enriched probe. The developed film shows no signal in the WT wells and one dominant signal in the transformed cells lines, confirming that the transformation vector had successfully recombined in a unique and single locus of the plastid without any unwarranted, illegitimate recombination with the genome or the plastid. The positive control is overexposed as the molar contribution of the cat sequence in the transformation vector is great deal higher than in the plastid

    Journal: Plant Molecular Biology

    Article Title: Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments

    doi: 10.1007/s11103-016-0554-8

    Figure Lengend Snippet: A schematic representation of a double homologues recombination event between transformation vector (pCCATCH) and chloroplast genome ( a ). The sequence of the cat CH gene, under control of the C. merolae chloroplast promoter P dna K, (marked as white, curved rectangle ) of the dna K gene, was integrated into the chloroplast molecule at the selected position between the rpl 32 - psb A genes. The solid black rectangles represent the algal chloroplast sequence, also present in the transformation vector, flanking the cat CH cassettes and functioning as facilitators of double homologues recombination. b Verification of the insertion of the cat CH cassette into the rpl 32- psb A region of the chloroplast molecule via PCR and subsequent agarose gel electrophoresis of the PCR products. The PCR reaction was performed on the total DNA, isolated from the wild type ( WT ) and stable chloroplast transformant lines (transformed by B biolistic bombardment or P PEG method). Two sets of primers were used: 5UTF-5UTR amplifying a 4612 bp long product (marked as x) and 3UTF-3UTR amplifying a 3655 bp long product (marked as y). These two PCR products encompass the regions between cat CH gene and fragments from beyond the upstream or downstream homologous region in the transformation vector. The presence of the cat CH was confirmed by a PCR reaction with catCHStuI-catCHKpnI primers pair amplifying a 672 bp long product (marked as z). To exclude the possibility of unwarranted, random integration of the vector or its continuous presence in the cell, a fragment of the plasmid ori region was amplified with the pABB20R-pABB20L primer pair, yielding a 3420 bp long product, only in case of the vector control, thereby confirming the absence of any residual plasmid (marked as v). M molecular marker. c Southern blot hybridization of PCR products generated by the 5UTF-5UTR and 3UTF-3UTR primer pairs with the CATCH probe (being the PCR product generated with catCHStuI-catCHKpnI primers pair). The total DNA, isolated from the stable chloroplast transformants with the integrated pCCATCH, obtained via biolistic bombardment ( B ) or PEG method ( P ) was used as a templates for PCR. The correct integration of the transformation vector was confirmed by the size of apparent bands (4612 bp and 3655 bp, see the upper part ) and additionally by Southern blot hybridization with cat probe (the lower part ). d The Southern blot hybridization of BglII-digested total DNA with the psb A probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with BglII restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 8 µg of DNA. The DNA was transferred onto a nitrocellulose membrane and hybridized with psbA probe. The transformant-derived psbA containing band was calculated to have the length of 4.2 kbp. The WT control fragment was calculated to form a 2.5 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared psb A probe. The semiquantitative results showed two bands (pointed out with arrows ) in the P and B lines, representing the wild type ptDNA band and 1.8 kbp larger (containing P dnaK cat CH cassette) recombined ptDNA band. The presence of both types of plastids demonstrated heterogeneity of the obtained transformant lineages. e The Southern blot hybridization of EcoRI-digested total DNA with the cat CH probe. The total DNA was isolated from both lineages ( P, B ) of the transformed cells as well as the wild type. After digesting with EcoRI restriction enzyme the total DNA was loaded onto the agarose gel in equal amounts of 5 µg of DNA. The cat containing band was calculated to have the length of 10 kbp. Additionally, a positive control was added by digesting the transformation plasmid with EcoRI and AatII. This fragment was calculated to range ~10 kbp band. The gel was transferred onto a nitrocellulose membrane and hybridized with an earlier prepared 32 P enriched probe. The developed film shows no signal in the WT wells and one dominant signal in the transformed cells lines, confirming that the transformation vector had successfully recombined in a unique and single locus of the plastid without any unwarranted, illegitimate recombination with the genome or the plastid. The positive control is overexposed as the molar contribution of the cat sequence in the transformation vector is great deal higher than in the plastid

    Article Snippet: Further, the DNA was digested with BglII restriction enzyme (Thermo, USA), cutting the plastid in the proximity of 3′ and 5′ ends of the psbA gene.

    Techniques: Transformation Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Amplification, Marker, Southern Blot, Hybridization, Generated, Derivative Assay, Positive Control

    Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific BglI restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. PCR products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.

    Journal: PLoS ONE

    Article Title: Elevated Expression of H19 and Igf2 in the Female Mouse Eye

    doi: 10.1371/journal.pone.0056611

    Figure Lengend Snippet: Sex-specific imprinting assay of H19 . A. RFLP experimental design. DNA sequencing of C57BL/6 and SD7 confirmed an SD7-specific BglI restriction site located within H19 exon 5. p11, p12 and p13 designate the locations of the RFLP primers listed in Table 1 . B. Confirmation of SD7-specificity of the BglI restriction. PCR products of H19 amplified from BL6 and SD7 gDNA (using primers p12 and p13) digested with BglI, and an undigested SD7 sample. C. Imprinting assay of H19 in male and female mice. PCR products of H19 amplified from eye cDNA derived from three F1 males (M1-3) and three F1 females (F1-3) of the ♂C57BL/6×♀SD7 cross (using primers p11 and p13) digested with BglI. Controls for the paternal (C57BL/6) and maternal (SD7) allele, and negative controls for the PCR are shown to the right.

    Article Snippet: 10 µl PCR product was incubated with 20 U BglI restriction enzyme (Fermentas), 3 µl Buffer O (Fermentas), ddH2 O to a total volume of 30 µl, 37°C; 28 h. Fragments were separated on a 2% agarose gel and stained with ethidium bromide.

    Techniques: DNA Sequencing, Polymerase Chain Reaction, Amplification, Mouse Assay, Derivative Assay

    Minicircle Donor Plasmid Generation to Improve HDR Efficiency (A) The MC-DP contained the homology COL7A1 arms for HDR, flanking the recognition sites for the restriction enzymes EcoRI and ClaI, a GFP IRES blasticidin cassette under the control of an EF1 promoter for an initial selection of transfected patient cells, and an introduced T > C silent mutation for HDR detection. (B) PCR analysis using a specific forward primer binding to exon 76 of COL7A1 and a specific reverse primer binding the integrated restriction sites within intron 80 revealed the correct integration of the restriction sites via HDR in spCas9/MC-DP-treated cells. Untreated RDEB patient keratinocytes (RDEB Kc) were included as negative control. (C) BglI digestion assay: PCR amplification of the COL7A1 target region, spanning from exon 76 to intron 80 of COL7A1 (1,040 bp), and subsequent BglI digestion revealed repair of the mutation in a part of blasticidin-selected cells, indicated by the obtained lower band at 957 bp upon digestion. The resulting digestion pattern of spCas9-C44 indicated a homozygous correction of the mutation. Positive control: hKc; negative control: RDEB Kc. (D) Sequence analysis of single-cell clone spCas9-C44 confirmed the correction of the InsC mutation within exon 80 on both alleles.

    Journal: Molecular Therapy

    Article Title: COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

    doi: 10.1016/j.ymthe.2017.07.005

    Figure Lengend Snippet: Minicircle Donor Plasmid Generation to Improve HDR Efficiency (A) The MC-DP contained the homology COL7A1 arms for HDR, flanking the recognition sites for the restriction enzymes EcoRI and ClaI, a GFP IRES blasticidin cassette under the control of an EF1 promoter for an initial selection of transfected patient cells, and an introduced T > C silent mutation for HDR detection. (B) PCR analysis using a specific forward primer binding to exon 76 of COL7A1 and a specific reverse primer binding the integrated restriction sites within intron 80 revealed the correct integration of the restriction sites via HDR in spCas9/MC-DP-treated cells. Untreated RDEB patient keratinocytes (RDEB Kc) were included as negative control. (C) BglI digestion assay: PCR amplification of the COL7A1 target region, spanning from exon 76 to intron 80 of COL7A1 (1,040 bp), and subsequent BglI digestion revealed repair of the mutation in a part of blasticidin-selected cells, indicated by the obtained lower band at 957 bp upon digestion. The resulting digestion pattern of spCas9-C44 indicated a homozygous correction of the mutation. Positive control: hKc; negative control: RDEB Kc. (D) Sequence analysis of single-cell clone spCas9-C44 confirmed the correction of the InsC mutation within exon 80 on both alleles.

    Article Snippet: For analysis of the repair efficiency via HDR, the target region was PCR-amplified using a COL7A1 exon 76-specific forward primer (5′-ATGAGCCAGGTCCTGGACTCTCTG-3′) and a COL7A1 intron 80-specific reverse primer (5′-GATCGGTACCTATGCGGCCGCGGGGCAGGGCACAGGATGGGGGC-3′) and subsequently digested with the restriction enzyme BglI (Thermo Fisher Scientific).

    Techniques: Plasmid Preparation, Selection, Transfection, Mutagenesis, Polymerase Chain Reaction, Binding Assay, Negative Control, Amplification, Positive Control, Sequencing

    HDR-Induced Correction of the COL7A1 Mutation 6527insC (A) PCR amplification of the COL7A1 -targeting region (1,190 bp) using primers binding exon 76 of COL7A1 and the DP sequence downstream of the 5′ homologous arm revealed the accurate integration of the selection cassette into intron 80 of COL7A1 exclusively in CRISPR/Cas9-treated RDEB patient cells. Negative controls: hKc, RDEB Kc, and DP. (B) PCR amplification of the COL7A1 target region spanning from exon 76 to intron 80 and subsequent BglI digestion assay revealed a digestion pattern of 957 and 83 bp (only upper digestion band shown), showing a partial correction of the mutation in both spCas9- and Cas9n-treated RDEB mixed-cell population and a heterozygous repair of single-cell clones spCas9-C21 and Cas9n-C28. Positive control: hKc; negative control: RDEB Kc. (C) Sequence analysis of the target site confirmed the correction of the mutations in CRISPR/Cas9-treated RDEB Kc, shown by the overlapping peaks, which correspond to guanine (wild-type [WT]) and cytosine (mutation [mut]) (red arrows). (D) Western blot analysis showed increased type VII collagen levels at 290 kDa in total cell lysates and cell culture supernatants taken from both single-cell clones spCas9-C21 and Cas9n-C28 compared with RDEB Kc. Positive control: hKc. Ponceau red and α-actinin staining were used as loading controls. C7: type VII collagen. (E) Immunofluorescence staining of type VII collagen expression revealed increased protein levels in both isolated single-cell clones spCas9-C21 and Cas9n-C28 compared with untreated RDEB Kc. Positive control: hKc. Cell nuclei were stained with DAPI. The scale bar represents 50 μm.

    Journal: Molecular Therapy

    Article Title: COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

    doi: 10.1016/j.ymthe.2017.07.005

    Figure Lengend Snippet: HDR-Induced Correction of the COL7A1 Mutation 6527insC (A) PCR amplification of the COL7A1 -targeting region (1,190 bp) using primers binding exon 76 of COL7A1 and the DP sequence downstream of the 5′ homologous arm revealed the accurate integration of the selection cassette into intron 80 of COL7A1 exclusively in CRISPR/Cas9-treated RDEB patient cells. Negative controls: hKc, RDEB Kc, and DP. (B) PCR amplification of the COL7A1 target region spanning from exon 76 to intron 80 and subsequent BglI digestion assay revealed a digestion pattern of 957 and 83 bp (only upper digestion band shown), showing a partial correction of the mutation in both spCas9- and Cas9n-treated RDEB mixed-cell population and a heterozygous repair of single-cell clones spCas9-C21 and Cas9n-C28. Positive control: hKc; negative control: RDEB Kc. (C) Sequence analysis of the target site confirmed the correction of the mutations in CRISPR/Cas9-treated RDEB Kc, shown by the overlapping peaks, which correspond to guanine (wild-type [WT]) and cytosine (mutation [mut]) (red arrows). (D) Western blot analysis showed increased type VII collagen levels at 290 kDa in total cell lysates and cell culture supernatants taken from both single-cell clones spCas9-C21 and Cas9n-C28 compared with RDEB Kc. Positive control: hKc. Ponceau red and α-actinin staining were used as loading controls. C7: type VII collagen. (E) Immunofluorescence staining of type VII collagen expression revealed increased protein levels in both isolated single-cell clones spCas9-C21 and Cas9n-C28 compared with untreated RDEB Kc. Positive control: hKc. Cell nuclei were stained with DAPI. The scale bar represents 50 μm.

    Article Snippet: For analysis of the repair efficiency via HDR, the target region was PCR-amplified using a COL7A1 exon 76-specific forward primer (5′-ATGAGCCAGGTCCTGGACTCTCTG-3′) and a COL7A1 intron 80-specific reverse primer (5′-GATCGGTACCTATGCGGCCGCGGGGCAGGGCACAGGATGGGGGC-3′) and subsequently digested with the restriction enzyme BglI (Thermo Fisher Scientific).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Binding Assay, Sequencing, Selection, CRISPR, Clone Assay, Positive Control, Negative Control, Western Blot, Cell Culture, Staining, Immunofluorescence, Expressing, Isolation