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    BsaI
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    BsaI 5 000 units
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    r0535l
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    Category:
    Restriction Enzymes
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    New England Biolabs bsa i
    BsaI
    BsaI 5 000 units
    https://www.bioz.com/result/bsa i/product/New England Biolabs
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    bsa i - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea"

    Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2014.29.9.1240

    Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Techniques Used: Mutagenesis, DNA Sequencing

    2) Product Images from "FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science"

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2015.172

    Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.
    Figure Legend Snippet: Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Techniques Used: Construct, Sequencing, Ligation, Binding Assay, Generated

    3) Product Images from "PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin"

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.4.1156-1157.2002

    Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).
    Figure Legend Snippet: Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Electrophoresis, Staining, Amplification

    4) Product Images from "Targeted mutagenesis in soybean using the CRISPR-Cas9 system"

    Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

    Journal: Scientific Reports

    doi: 10.1038/srep10342

    Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.
    Figure Legend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Techniques Used: Derivative Assay

    5) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    6) Product Images from "Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem"

    Article Title: Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.16.8650-8660.2003

    Relative DNA replication under one-step growth conditions. (A and C) DNA extracted from single-cycle infections using wild-type and no-cre viruses in A9 (A) and 324K (C) cells, with autoradiographs positioned above the gels from which they were created by Southern blotting. Individual lanes are aligned and contain total DNA extracted at 12, 24, and 36 h postinfection and then digested with Bsa I. DNA extracted from equivalent amounts of both cells and medium are shown for each time point. The gels were stained with ethidium bromide, and the position of the double-stranded (ds) 5-kb band is shown. (B and D) On the Southern blots, the positions of 10-kb and 5-kb viral double-stranded bands, as well as those of 5-kb single-stranded (ss) progeny genomes, are indicated. Results for all five viruses, tested in A9 (B) and 324K (D) cells, are expressed in graphic form as the quantity of viral DNA (in nanograms), normalized to the amount of total DNA (in micrograms) in each sample. Each bar represents three independent experiments, with the standard deviations indicated. The numbers under each bar of the graph indicate hours postinfection.
    Figure Legend Snippet: Relative DNA replication under one-step growth conditions. (A and C) DNA extracted from single-cycle infections using wild-type and no-cre viruses in A9 (A) and 324K (C) cells, with autoradiographs positioned above the gels from which they were created by Southern blotting. Individual lanes are aligned and contain total DNA extracted at 12, 24, and 36 h postinfection and then digested with Bsa I. DNA extracted from equivalent amounts of both cells and medium are shown for each time point. The gels were stained with ethidium bromide, and the position of the double-stranded (ds) 5-kb band is shown. (B and D) On the Southern blots, the positions of 10-kb and 5-kb viral double-stranded bands, as well as those of 5-kb single-stranded (ss) progeny genomes, are indicated. Results for all five viruses, tested in A9 (B) and 324K (D) cells, are expressed in graphic form as the quantity of viral DNA (in nanograms), normalized to the amount of total DNA (in micrograms) in each sample. Each bar represents three independent experiments, with the standard deviations indicated. The numbers under each bar of the graph indicate hours postinfection.

    Techniques Used: Southern Blot, Staining

    The MVM-Chop system for generating mutations in the left-hand terminal hairpin. (A) A map of pChopII, indicating the two Bsa I restriction sites, is shown in the upper left, where gray indicates MVM sequence and black indicates bacterial vector. Digestion with Bsa I and subsequent gel purification produced a 6-kb linear DNA with noncohesive 5′ overhangs at each end. Synthetic oligonucleotides that regenerate the terminal hairpin were ligated to the linearized plasmid. This construct was transfected into permissive cells from which mutant virus could be plaque purified for future analysis. (B) The sequence of the wild-type oligonucleotide is shown in hairpin form, with the two PIF half-sites and the CRE indicated by open and shaded boxes, respectively. The arrow with an asterisk indicates the identification of an A residue which differs from the previously published MVMp sequence, as discussed in the text. The sequences of the central regions of mutant hairpin oligonucleotides used in this study are shown below the wild-type hairpin. Altered and deleted sequences are indicated by gray boxes and arrows, respectively. Ellipses (…) indicate continuation of the wild-type sequence.
    Figure Legend Snippet: The MVM-Chop system for generating mutations in the left-hand terminal hairpin. (A) A map of pChopII, indicating the two Bsa I restriction sites, is shown in the upper left, where gray indicates MVM sequence and black indicates bacterial vector. Digestion with Bsa I and subsequent gel purification produced a 6-kb linear DNA with noncohesive 5′ overhangs at each end. Synthetic oligonucleotides that regenerate the terminal hairpin were ligated to the linearized plasmid. This construct was transfected into permissive cells from which mutant virus could be plaque purified for future analysis. (B) The sequence of the wild-type oligonucleotide is shown in hairpin form, with the two PIF half-sites and the CRE indicated by open and shaded boxes, respectively. The arrow with an asterisk indicates the identification of an A residue which differs from the previously published MVMp sequence, as discussed in the text. The sequences of the central regions of mutant hairpin oligonucleotides used in this study are shown below the wild-type hairpin. Altered and deleted sequences are indicated by gray boxes and arrows, respectively. Ellipses (…) indicate continuation of the wild-type sequence.

    Techniques Used: Sequencing, Plasmid Preparation, Gel Purification, Produced, Construct, Transfection, Mutagenesis, Purification

    7) Product Images from "Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1 [OPEN]"

    Article Title: Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.18.00200

    Construction of the sgRNA/Cas9 system. A, Partial sequence alignment of the k1C gene family from the reference and sgRNA design. The gray line at the bottom indicates a PAM; the black line shows a 19-nucleotide consensus DNA target region. sgRNA is complementary to the DNA target region. B, Schematic diagram of the construction of the sgRNA/Cas9 system. Gray arrows indicate the cohesive end produced by Bsa I digestion in the vector. The synthetic double-stranded sgRNA with four-nucleotide 5′ overhangs at both ends was inserted into the sgRNA/Cas9 expression system. The black triangle represents a G base in the TaU3 promoter followed by the 5′ end of the 19-nucleotide sgRNA that resulted in a final 20-bp sgRNA with G (N19-nt) in the guide RNA/Cas9 system and introduced an artificial mismatch in the target site of the k1C members. RB/LB represent the left and right borders of the vector; TaU3P is the wheat U3 gene promoter; the gRNA site refers to the guide RNA clone site; gRNA SC is the gRNA scaffold; Ubi P is the Ubiquitin gene promoter; Zm Cas9 is the maize codon-optimized Cas9 ; Nos t is the Nos terminator; 35S P is the 35S promoter; Npt II is the neomycin phosphotransferase II gene.
    Figure Legend Snippet: Construction of the sgRNA/Cas9 system. A, Partial sequence alignment of the k1C gene family from the reference and sgRNA design. The gray line at the bottom indicates a PAM; the black line shows a 19-nucleotide consensus DNA target region. sgRNA is complementary to the DNA target region. B, Schematic diagram of the construction of the sgRNA/Cas9 system. Gray arrows indicate the cohesive end produced by Bsa I digestion in the vector. The synthetic double-stranded sgRNA with four-nucleotide 5′ overhangs at both ends was inserted into the sgRNA/Cas9 expression system. The black triangle represents a G base in the TaU3 promoter followed by the 5′ end of the 19-nucleotide sgRNA that resulted in a final 20-bp sgRNA with G (N19-nt) in the guide RNA/Cas9 system and introduced an artificial mismatch in the target site of the k1C members. RB/LB represent the left and right borders of the vector; TaU3P is the wheat U3 gene promoter; the gRNA site refers to the guide RNA clone site; gRNA SC is the gRNA scaffold; Ubi P is the Ubiquitin gene promoter; Zm Cas9 is the maize codon-optimized Cas9 ; Nos t is the Nos terminator; 35S P is the 35S promoter; Npt II is the neomycin phosphotransferase II gene.

    Techniques Used: Sequencing, Produced, Plasmid Preparation, Expressing

    8) Product Images from "A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice"

    Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

    Journal: PeerJ

    doi: 10.7717/peerj.8491

    Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.
    Figure Legend Snippet: Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

    Techniques Used: Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Incubation

    Construction of the destination vectors PJG097 without Bas I sites. There are two BsaI sites in the backbone of pH-Ubi-cas9-7. To remove the BsaI sites, primers containing site mutation in its recognition site was designed (referred to as OJK121 and OJK122). The Bsa I sites and matched adaptors were added to the 5′ stream of primers. An amplification with OJK121 and OJK122 was conducted with pH-Ubi-cas9-7 as the template. Gel-purified PCR product and pH-Ubi-cas9-7 was then digested with BsaI and then submitted to a ligation reaction. The product was then transferred into DB3.1 competent cells (ZOMANBIO Co., Shanghai, China). Positive clones were then verified by DNA sequencing and digestion with BsaI. The new vector removing BsaI sites was renamed as PJG097. An LR reaction was performed with 30 ng pOs-sgRNA and PJG097, producing the destination clone named PJG112. PJG112 could serve as destination vector to construct expression clones containing two spacers.
    Figure Legend Snippet: Construction of the destination vectors PJG097 without Bas I sites. There are two BsaI sites in the backbone of pH-Ubi-cas9-7. To remove the BsaI sites, primers containing site mutation in its recognition site was designed (referred to as OJK121 and OJK122). The Bsa I sites and matched adaptors were added to the 5′ stream of primers. An amplification with OJK121 and OJK122 was conducted with pH-Ubi-cas9-7 as the template. Gel-purified PCR product and pH-Ubi-cas9-7 was then digested with BsaI and then submitted to a ligation reaction. The product was then transferred into DB3.1 competent cells (ZOMANBIO Co., Shanghai, China). Positive clones were then verified by DNA sequencing and digestion with BsaI. The new vector removing BsaI sites was renamed as PJG097. An LR reaction was performed with 30 ng pOs-sgRNA and PJG097, producing the destination clone named PJG112. PJG112 could serve as destination vector to construct expression clones containing two spacers.

    Techniques Used: Mutagenesis, Amplification, Purification, Polymerase Chain Reaction, Ligation, Clone Assay, DNA Sequencing, Plasmid Preparation, Construct, Expressing

    9) Product Images from "Transcriptional Activators of Human Genes with Programmable DNA-Specificity"

    Article Title: Transcriptional Activators of Human Genes with Programmable DNA-Specificity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019509

    Golden TAL Technology for assembly of TAL proteins with programmed repeat composition. (A) Single TAL repeats were cloned with flanking Bpi I sites that generate specific four base pair-overhangs (a–g). Matching sites are indicated by identical letters. A library was constructed with four different repeat types (RVD, repeat variable di-residue: NI, HD, NN, NG) for each repeat position. The repeat types have different DNA-specificities (Spec., only upper DNA-strand is shown). (B) One to six repeats are assembled in specific order into an assembly vector to generate a repeat assembly with flanking Bsa I sites. (C) TALs were directly assembled with N-terminal GFP-tag into an expression vector using fragments with matching Bsa I-generated overhangs (capital letters). Insertion of one to four repeat assemblies generated TALs with 1 to 24 repeats. The last repeat is only a half repeat as typical for TAL proteins. Please see the Text S1 and Figure S4 for details.
    Figure Legend Snippet: Golden TAL Technology for assembly of TAL proteins with programmed repeat composition. (A) Single TAL repeats were cloned with flanking Bpi I sites that generate specific four base pair-overhangs (a–g). Matching sites are indicated by identical letters. A library was constructed with four different repeat types (RVD, repeat variable di-residue: NI, HD, NN, NG) for each repeat position. The repeat types have different DNA-specificities (Spec., only upper DNA-strand is shown). (B) One to six repeats are assembled in specific order into an assembly vector to generate a repeat assembly with flanking Bsa I sites. (C) TALs were directly assembled with N-terminal GFP-tag into an expression vector using fragments with matching Bsa I-generated overhangs (capital letters). Insertion of one to four repeat assemblies generated TALs with 1 to 24 repeats. The last repeat is only a half repeat as typical for TAL proteins. Please see the Text S1 and Figure S4 for details.

    Techniques Used: Clone Assay, Construct, Plasmid Preparation, Expressing, Generated

    10) Product Images from "A CRISPR/Cas9 toolkit for multiplex genome editing in plants"

    Article Title: A CRISPR/Cas9 toolkit for multiplex genome editing in plants

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0327-y

    Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two Bsa I sites (not shown).
    Figure Legend Snippet: Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two Bsa I sites (not shown).

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction

    Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated Bsa I site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli ; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium . Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9 , Zea mays codon-optimized Cas9 ; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.
    Figure Legend Snippet: Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated Bsa I site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli ; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium . Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9 , Zea mays codon-optimized Cas9 ; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.

    Techniques Used: CRISPR, Derivative Assay, Plasmid Preparation, SPR Assay, Sequencing

    11) Product Images from "TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines 1"

    Article Title: TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines 1

    Journal: Plant Physiology

    doi: 10.1104/pp.15.00481

    Somatic mutagenesis and inheritance of TALEN-mediated mutations in CYP71A12 . A, Schematic representation of the analysis of targeted mutagenesis; the TALEN-binding site targeting one of two Bsa analysis for a wild-type plant and a homozygous mutant plant. C and D, Analysis of TALEN-induced mutations in CYP71A12 . The sequence of the CYP71A12 wild-type allele with TALEN-binding sites is in italic letters, and the targeted Bsa I restriction site is in boldface; insertions are in lowercase letters, and deletions are indicated as dashes. C, Somatic events detected. D, Stable lines generated. prim. transf., Primary transformant; *, of the 82-bp deletion event, only 56 deleted bp are indicated.
    Figure Legend Snippet: Somatic mutagenesis and inheritance of TALEN-mediated mutations in CYP71A12 . A, Schematic representation of the analysis of targeted mutagenesis; the TALEN-binding site targeting one of two Bsa analysis for a wild-type plant and a homozygous mutant plant. C and D, Analysis of TALEN-induced mutations in CYP71A12 . The sequence of the CYP71A12 wild-type allele with TALEN-binding sites is in italic letters, and the targeted Bsa I restriction site is in boldface; insertions are in lowercase letters, and deletions are indicated as dashes. C, Somatic events detected. D, Stable lines generated. prim. transf., Primary transformant; *, of the 82-bp deletion event, only 56 deleted bp are indicated.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing, Generated

    12) Product Images from "Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance"

    Article Title: Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.3.1167-1172.2003

    Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). Bsa I (lanes 1 to 5) or Bbs I (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). Bbs I digestion of the 521-bp fragment of additional isolates of B. pertussis . Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.
    Figure Legend Snippet: Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). Bsa I (lanes 1 to 5) or Bbs I (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). Bbs I digestion of the 521-bp fragment of additional isolates of B. pertussis . Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification

    13) Product Images from "GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris"

    Article Title: GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

    Journal: BMC Systems Biology

    doi: 10.1186/s12918-017-0492-3

    Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )
    Figure Legend Snippet: Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Construct, Expressing

    14) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    15) Product Images from "PCR Using 3?-Mismatched Primers To Detect A2142C Mutation in 23S rRNA Conferring Resistance to Clarithromycin in Helicobacter pylori Clinical Isolates"

    Article Title: PCR Using 3?-Mismatched Primers To Detect A2142C Mutation in 23S rRNA Conferring Resistance to Clarithromycin in Helicobacter pylori Clinical Isolates

    Journal: Journal of Clinical Microbiology

    doi:

    PCR-RFLP patterns obtained after digestion with Bsa I or Mbo II. Bsa I digested the 1.4-kbp fragment, producing 1,000- and 400-bp fragments in either susceptible or resistant strains. However, if the A2143G mutation was present, the 1,000-bp fragment was converted to 700- and 300-bp fragments. Mbo II digested the fragment, producing two fragments of 700 bp only when the A2142G mutation was present. Lanes: 1 and 2, strain 1 digested with Bsa I and Mbo II, respectively; 3 and 4, strain 2 digested with Bsa I and Mbo II, respectively; 5 and 6, strain 3 digested with Bsa I and Mbo II, respectively; 7 and 8, strain 4 digested with Bsa I and Mbo II, respectively; 9, DNA markers. Bsa I digested the fragment in strains 1 and 2. Bsa I or Mbo II did not digest strains 3 and 4.
    Figure Legend Snippet: PCR-RFLP patterns obtained after digestion with Bsa I or Mbo II. Bsa I digested the 1.4-kbp fragment, producing 1,000- and 400-bp fragments in either susceptible or resistant strains. However, if the A2143G mutation was present, the 1,000-bp fragment was converted to 700- and 300-bp fragments. Mbo II digested the fragment, producing two fragments of 700 bp only when the A2142G mutation was present. Lanes: 1 and 2, strain 1 digested with Bsa I and Mbo II, respectively; 3 and 4, strain 2 digested with Bsa I and Mbo II, respectively; 5 and 6, strain 3 digested with Bsa I and Mbo II, respectively; 7 and 8, strain 4 digested with Bsa I and Mbo II, respectively; 9, DNA markers. Bsa I digested the fragment in strains 1 and 2. Bsa I or Mbo II did not digest strains 3 and 4.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis

    16) Product Images from "Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe"

    Article Title: Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    Journal: Open Biology

    doi: 10.1098/rsob.150054

    The Golden Gate method to create integration plasmids. ( a ) A schematic of an integration plasmid. (left) An example of an integration plasmid to express a GOI in fusion with a fluorescent protein (FP) tag under a promoter. A target module comprises tar.F and tar.R regions separated by an Fse I restriction site. (right) An integration plasmid can be linearized with Fse I, and tar.F and tar.R sequences are targeted to the homologous sequences on the S. pombe chromosome, to induce homologous recombination. ( b ) A schematic for the Golden Gate reaction. (left) Examples of module elements. Modules are given either as plasmids (1, 3–6) or as PCR products (2). Modules for a promoter module (1), GOI (2), an FPtag (3), a selection marker (4), a target region (5), the vector backbone (6). a–f: cohesive ends to connect modules 1–6 in this order. (right) A reaction protocol for the Golden Gate reaction by the mixture of 1–6 and the resulting circular integration plasmid (7). Each module plasmid (1, 3–6) contains the kanamycin resistance gene (KanR), whereas the final product (integration plasmid, 7) is ampicillin resistant. ( c ) Unique property of Bsa I. (left) Eco RI, a standard restriction enzyme, cleaves its recognition site, therefore digestion and religation can be repeated. (right) By contrast, Bsa I has separate sites for recognition (GGTCTC) and digestion (NNNN; any four bases).
    Figure Legend Snippet: The Golden Gate method to create integration plasmids. ( a ) A schematic of an integration plasmid. (left) An example of an integration plasmid to express a GOI in fusion with a fluorescent protein (FP) tag under a promoter. A target module comprises tar.F and tar.R regions separated by an Fse I restriction site. (right) An integration plasmid can be linearized with Fse I, and tar.F and tar.R sequences are targeted to the homologous sequences on the S. pombe chromosome, to induce homologous recombination. ( b ) A schematic for the Golden Gate reaction. (left) Examples of module elements. Modules are given either as plasmids (1, 3–6) or as PCR products (2). Modules for a promoter module (1), GOI (2), an FPtag (3), a selection marker (4), a target region (5), the vector backbone (6). a–f: cohesive ends to connect modules 1–6 in this order. (right) A reaction protocol for the Golden Gate reaction by the mixture of 1–6 and the resulting circular integration plasmid (7). Each module plasmid (1, 3–6) contains the kanamycin resistance gene (KanR), whereas the final product (integration plasmid, 7) is ampicillin resistant. ( c ) Unique property of Bsa I. (left) Eco RI, a standard restriction enzyme, cleaves its recognition site, therefore digestion and religation can be repeated. (right) By contrast, Bsa I has separate sites for recognition (GGTCTC) and digestion (NNNN; any four bases).

    Techniques Used: Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Selection, Marker

    Choice of module plasmids for expression of a C-terminal tagged GOI. ( a ) Detailed illustration of an integration plasmid for expression of the GOI–GFP fusion (C-terminal GFP tag). Modules I–V are connected in the pFA6a-based vector (module VI) in that order. In this example, the adh1 promoter (selected from group I modules) drives expression of the fusion gene of the GOI (GOI (bc), II) with GFP (FPtag-C (cd), III). T adh serves as a terminator. kan (P TEF , promoter; T TEF , terminator) is a selection marker used after S. pombe transformation (module IV). Target module (V) is the sequence that is targeted to a homologous sequence in S. pombe chromosomes. Useful restriction sites are also indicated. Digestion with Not I separates the vector and other modules. JB19F and JB20R correspond to sequences commonly used in PCR-based gene targeting [ 12 ]. a–f in module names indicate the names of Bsa I cohesive ends used therein ( c ). AmpR, the ampicillin resistance gene. ( b , c ) List of module plasmids created in this study. ( b ) Modules are categorized as groups I–VI in boxes. Choose one module from each group to mix. II. The GOI (bc) is made through PCR to add cohesive ends (‘b’ and ‘c’). Group IIIa, instead of II and III, can be used to make control strains. Modules for the adh terminator and a selection marker gene can be supplied together (group IV). Alternatively, each module can be chosen separately: a terminator (group IVa) and a selection marker (group IVb). ( c ) Sequences of cohesive ends named a–g. Note that the module vector pBMod contains a Bsa I site with the cohesive end GTTA.
    Figure Legend Snippet: Choice of module plasmids for expression of a C-terminal tagged GOI. ( a ) Detailed illustration of an integration plasmid for expression of the GOI–GFP fusion (C-terminal GFP tag). Modules I–V are connected in the pFA6a-based vector (module VI) in that order. In this example, the adh1 promoter (selected from group I modules) drives expression of the fusion gene of the GOI (GOI (bc), II) with GFP (FPtag-C (cd), III). T adh serves as a terminator. kan (P TEF , promoter; T TEF , terminator) is a selection marker used after S. pombe transformation (module IV). Target module (V) is the sequence that is targeted to a homologous sequence in S. pombe chromosomes. Useful restriction sites are also indicated. Digestion with Not I separates the vector and other modules. JB19F and JB20R correspond to sequences commonly used in PCR-based gene targeting [ 12 ]. a–f in module names indicate the names of Bsa I cohesive ends used therein ( c ). AmpR, the ampicillin resistance gene. ( b , c ) List of module plasmids created in this study. ( b ) Modules are categorized as groups I–VI in boxes. Choose one module from each group to mix. II. The GOI (bc) is made through PCR to add cohesive ends (‘b’ and ‘c’). Group IIIa, instead of II and III, can be used to make control strains. Modules for the adh terminator and a selection marker gene can be supplied together (group IV). Alternatively, each module can be chosen separately: a terminator (group IVa) and a selection marker (group IVb). ( c ) Sequences of cohesive ends named a–g. Note that the module vector pBMod contains a Bsa I site with the cohesive end GTTA.

    Techniques Used: Expressing, Plasmid Preparation, Selection, Marker, Transformation Assay, Sequencing, Polymerase Chain Reaction

    17) Product Images from "A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae"

    Article Title: A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.26309

    Scheme of the plug-and-play pathway refactoring workflow. (A) The 1 st tier Golden Gate reaction. The gene is either synthesized or PCR amplified with Bbs I cleavage sites at both ends and cloned into a helper plasmid through Bbs I catalyzed Golden Gate reaction. (B) The 2 nd tier Golden Gate reaction. Helper plasmids harboring the corresponding genes are mixed with the appropriate spacer plasmids and the receiver plasmid and assembled into the final construct through Bsa I catalyzed Golden Gate reaction. All helper plasmids and spacer plasmids share the pUC19 backbone, while the receiver plasmid has either a pET28a backbone (for expression in E. coli ) or pRS416 backbone with the ampicillin resistance gene replaced by the kanamycin resistance gene (for expression in S. cerevisiae ).
    Figure Legend Snippet: Scheme of the plug-and-play pathway refactoring workflow. (A) The 1 st tier Golden Gate reaction. The gene is either synthesized or PCR amplified with Bbs I cleavage sites at both ends and cloned into a helper plasmid through Bbs I catalyzed Golden Gate reaction. (B) The 2 nd tier Golden Gate reaction. Helper plasmids harboring the corresponding genes are mixed with the appropriate spacer plasmids and the receiver plasmid and assembled into the final construct through Bsa I catalyzed Golden Gate reaction. All helper plasmids and spacer plasmids share the pUC19 backbone, while the receiver plasmid has either a pET28a backbone (for expression in E. coli ) or pRS416 backbone with the ampicillin resistance gene replaced by the kanamycin resistance gene (for expression in S. cerevisiae ).

    Techniques Used: Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Construct, Expressing

    18) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    19) Product Images from "Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha"

    Article Title: Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha

    Journal: bioRxiv

    doi: 10.1101/2020.02.29.971002

    Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.
    Figure Legend Snippet: Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.

    Techniques Used: Sequencing, Plasmid Preparation, Transformation Assay, Construct, CRISPR

    20) Product Images from "New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1 [W]New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1 [W] [OPEN]"

    Article Title: New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1 [W]New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis 1 [W] [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.113.234989

    s in vectors containing a modified version of AtTAS1c with a ccd B cassette flanked by two Bsa I sites (B/c vectors). A, Diagram of AtTAS1c production is initiated by miR173-guided cleavage
    Figure Legend Snippet: s in vectors containing a modified version of AtTAS1c with a ccd B cassette flanked by two Bsa I sites (B/c vectors). A, Diagram of AtTAS1c production is initiated by miR173-guided cleavage

    Techniques Used: Modification

    21) Product Images from "DNA end-joining catalyzed by human cell-free extracts"

    Article Title: DNA end-joining catalyzed by human cell-free extracts

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    DNA end-joining catalyzed by human cell-free extracts. ( A ) Protein extracts from GM00558 were incubated with 5′- 32 P-end-labeled Bsa I-linearized pDEA-7Z DNA as described in Materials and Methods (lanes 1–4). Lane 5, DNA ligation ladder. ( B ) Extracts of three lymphoblastoid cell lines were analyzed for their ability to promote end-joining under standard assay conditions. ( C ) End-joining catalyzed by GM00558 extract (68 μg) was analyzed by using uniformly 32 P-labeled pFB585 DNA (100 ng) linearized with Bsa I (lanes 1 and 2), Eco RV (lane 3), or Kpn I (lane 4). ( D ) Ligation products from reactions similar to those shown in C were purified by gel electrophoresis and treated with (+) or without (−) Bsa I (lanes 1 and 2), Eco RV (lanes 3 and 4), or Kpn I (lanes 5 and 6).
    Figure Legend Snippet: DNA end-joining catalyzed by human cell-free extracts. ( A ) Protein extracts from GM00558 were incubated with 5′- 32 P-end-labeled Bsa I-linearized pDEA-7Z DNA as described in Materials and Methods (lanes 1–4). Lane 5, DNA ligation ladder. ( B ) Extracts of three lymphoblastoid cell lines were analyzed for their ability to promote end-joining under standard assay conditions. ( C ) End-joining catalyzed by GM00558 extract (68 μg) was analyzed by using uniformly 32 P-labeled pFB585 DNA (100 ng) linearized with Bsa I (lanes 1 and 2), Eco RV (lane 3), or Kpn I (lane 4). ( D ) Ligation products from reactions similar to those shown in C were purified by gel electrophoresis and treated with (+) or without (−) Bsa I (lanes 1 and 2), Eco RV (lanes 3 and 4), or Kpn I (lanes 5 and 6).

    Techniques Used: Incubation, Labeling, DNA Ligation, Ligation, Purification, Nucleic Acid Electrophoresis

    22) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    Related Articles

    Mutagenesis:

    Article Title: Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
    Article Snippet: .. BsaI and BpiI sites were removed in a so-called “domestication” procedure using a Q5 site-directed mutagenesis (SDM) kit (NEB). ..

    Polymerase Chain Reaction:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
    Article Snippet: .. 160 ng of pooled VH PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs). .. After cloning, six reactions were pooled, purified using Wizard® SV Gel and PCR Clean-up System (Promega) and eluted in a final volume of 30 µL which were subsequently used for one electroporation reaction into EBY100 as previously described by Benatuil et al. [ ].

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin
    Article Snippet: .. The 267-bp PCR products were precipitated and suspended in 15 μl of H2 O, and 5 μl was digested overnight in a final volume of 15 μl with the restriction enzymes Bbs I (5 U), Bsa I (5 U) , and Bce AI (0.5 U) (New England Biolabs). .. PCR-RFLP allowed the identification of mutations A2142G and A2143G using the Bbs I and Bsa I restriction enzymes, respectively (Fig. , lanes 3 and 5), as previously described ( , , ).

    Expressing:

    Article Title: GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system
    Article Snippet: .. During another especially challenging project where 15 expression cassettes were to be assembled, we compared the influence on efficiency of two BsaI enzyme variants (BsaI from New England Biolabs Cat. No. R0535S and BsaI-HFv2 also from NEB Cat. No. R3733S). .. The BsaI-HFv2 has greatly enhanced the efficiency of more challenging assemblies.

    Plasmid Preparation:

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: .. Briefly, for each reaction, 25 ng of each plasmid (for trimer libraries, 13 plasmids; for dimer libraries, 9 plasmids) ( ) was combined with 10 U of Bsa I (New England BioLabs, Ipswich, MA) and 400 U of T4 DNA ligase (New England BioLabs) in 1× T4 DNA ligase buffer (reaction volume, 20 μl). .. Three digest–ligation cycles were performed (10-min digest at 37°C; 15-min ligation at 16°C), followed by two 5-min inactivation steps at 50°C and 80°C.

    Purification:

    Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system
    Article Snippet: .. These two plasmids were digested completely using Bsa I (NEB, Massachusetts, USA) and purified with a TIANquick Midi purification kit (Tiangen, Beijing, China). ..

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    New England Biolabs bsa i
    Restriction fragment length polymorphism analysis of <t>23S</t> rRNA amplicons: ( A ) digestion with <t>Bsa</t> I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.
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    Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Journal: Journal of Korean Medical Science

    Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

    doi: 10.3346/jkms.2014.29.9.1240

    Figure Lengend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Article Snippet: Amplicons (424 bp each) of the 23S rRNA gene were digested with either Bsa I (New England BioLabs, Beverly, MA, USA) for 14 hr at 50℃ or Bbs I (New England BioLabs) for 14 hr at 37℃ to detect the A2144G and A2143G mutations, respectively ( ).

    Techniques: Mutagenesis, DNA Sequencing

    Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Journal: Human Gene Therapy

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    doi: 10.1089/hum.2015.172

    Figure Lengend Snippet: Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Article Snippet: Briefly, for each reaction, 25 ng of each plasmid (for trimer libraries, 13 plasmids; for dimer libraries, 9 plasmids) ( ) was combined with 10 U of Bsa I (New England BioLabs, Ipswich, MA) and 400 U of T4 DNA ligase (New England BioLabs) in 1× T4 DNA ligase buffer (reaction volume, 20 μl).

    Techniques: Construct, Sequencing, Ligation, Binding Assay, Generated

    Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin

    doi: 10.1128/AAC.46.4.1156-1157.2002

    Figure Lengend Snippet: Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Article Snippet: The 267-bp PCR products were precipitated and suspended in 15 μl of H2 O, and 5 μl was digested overnight in a final volume of 15 μl with the restriction enzymes Bbs I (5 U), Bsa I (5 U) , and Bce AI (0.5 U) (New England Biolabs).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Electrophoresis, Staining, Amplification

    Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Journal: Scientific Reports

    Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

    doi: 10.1038/srep10342

    Figure Lengend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Article Snippet: These two plasmids were digested completely using Bsa I (NEB, Massachusetts, USA) and purified with a TIANquick Midi purification kit (Tiangen, Beijing, China).

    Techniques: Derivative Assay