bsai New England Biolabs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs bsai
    Incorporation of double and single BrdU residues by <t>Bst</t> exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, <t>BsaI-cut</t> 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.
    Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsai/product/New England Biolabs
    Average 99 stars, based on 1561 article reviews
    Price from $9.99 to $1999.99
    bsai - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs m7g 50 ppp 50 g rna cap structure analog new england biolabs
    Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked <t>pUC19;</t> li = <t>BsaI-HF</t> linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.
    M7g 50 Ppp 50 G Rna Cap Structure Analog New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m7g 50 ppp 50 g rna cap structure analog new england biolabs/product/New England Biolabs
    Average 99 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    m7g 50 ppp 50 g rna cap structure analog new england biolabs - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs bsaihfv2
    Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked <t>pUC19;</t> li = <t>BsaI-HF</t> linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.
    Bsaihfv2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsaihfv2/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    bsaihfv2 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs endonuclease n bsai
    Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked <t>pUC19;</t> li = <t>BsaI-HF</t> linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.
    Endonuclease N Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/endonuclease n bsai/product/New England Biolabs
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    endonuclease n bsai - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Journal: BMC Biochemistry

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    doi: 10.1186/1471-2091-12-47

    Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

    Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.

    Journal: BMC Biochemistry

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    doi: 10.1186/1471-2091-12-47

    Figure Lengend Snippet: Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.

    Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

    Techniques: Polymerase Chain Reaction

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.

    Journal: BMC Biochemistry

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    doi: 10.1186/1471-2091-12-47

    Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.

    Article Snippet: Bst DNA Polymerase large fragment exo-, pUC19 DNA, BsaI, NcoI and BspHI, T4 DNA ligase REase were from New England Biolabs (Ipswich, MA, USA).

    Techniques: Polymerase Chain Reaction, Functional Assay

    Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones

    Journal: BMC Biotechnology

    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

    doi: 10.1186/s12896-020-00616-z

    Figure Lengend Snippet: Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones

    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).

    Techniques: Clone Assay, Agarose Gel Electrophoresis

    Chromatograms of PCR-RFLP assays and sequencing for detection of nucleotide alterations of 23S rRNA . H. Pylori 26695 and CLR r -1 were used as negative and positive control of A2143G mutation. BsaI digestion of the PCR products of representative samples was displayed on 8% PAGE gel. The 289 bp A2143G-positive PCR products were cleaved into a 199 bp and a 90 bp fragments ( A ). The A2143G mutation was also confirmed by sequencing of the PCR products of 23S rRNA ( B , displayed 2140–2154 fragment). H. Pylori 26695 and a 2142G clone were used as negative and positive control of A2142G mutation. MboII digestion of the PCR products of representative samples was displayed on 2% agarose gel. The 289 bp A2142G-positive PCR products of 2142G were cleaved into an 182 bp and a 107 bp fragments. The PCR product of GJ2040 was cleaved into a 164 bp and a 125 bp fragments; and the product of GJ2111 was cleaved into 245 bp and 44 bp fragment(s) ( C ). The A2142G and other mutations were confirmed by sequencing ( D ). Two new MboII -sensitive sequences were characterized as CTTCA (2222–2226) for GJ2040 and GAAG (2081–2084) for GJ2111.

    Journal: BMC Microbiology

    Article Title: Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history

    doi: 10.1186/1471-2180-8-81

    Figure Lengend Snippet: Chromatograms of PCR-RFLP assays and sequencing for detection of nucleotide alterations of 23S rRNA . H. Pylori 26695 and CLR r -1 were used as negative and positive control of A2143G mutation. BsaI digestion of the PCR products of representative samples was displayed on 8% PAGE gel. The 289 bp A2143G-positive PCR products were cleaved into a 199 bp and a 90 bp fragments ( A ). The A2143G mutation was also confirmed by sequencing of the PCR products of 23S rRNA ( B , displayed 2140–2154 fragment). H. Pylori 26695 and a 2142G clone were used as negative and positive control of A2142G mutation. MboII digestion of the PCR products of representative samples was displayed on 2% agarose gel. The 289 bp A2142G-positive PCR products of 2142G were cleaved into an 182 bp and a 107 bp fragments. The PCR product of GJ2040 was cleaved into a 164 bp and a 125 bp fragments; and the product of GJ2111 was cleaved into 245 bp and 44 bp fragment(s) ( C ). The A2142G and other mutations were confirmed by sequencing ( D ). Two new MboII -sensitive sequences were characterized as CTTCA (2222–2226) for GJ2040 and GAAG (2081–2084) for GJ2111.

    Article Snippet: RFLP assays The 289 bp amplicon of 23S rRNA was digested with the restriction enzymes BsaI and MboII (New England Biolabs, USA) in order to detect A2143G and A2142G point mutations, respectively (Fig ) [ , ].

    Techniques: Polymerase Chain Reaction, Sequencing, Positive Control, Mutagenesis, Polyacrylamide Gel Electrophoresis, Agarose Gel Electrophoresis

    Assembly of a GFP construct from 10 plasmids. (A) Construct maps. Input modules contain a core region C flanked by BsaI restriction sites in opposite orientation composed of a recognition site (B, ggtctcn, B , ngagacc) and a 4 nucleotide cleavage site (boxes flanking the core region). pX-LacZ, acceptor vector. pGFPi, resulting construct. Restriction sites for AvrII and XmaI are shown as white arrows. (B) Ethidium bromide-stained gel with products obtained by restriction-ligation of the 9 input module plasmids. M: GeneRuler 1kb DNA Ladder Plus from Fermentas. Restriction-ligation was performed at 37°C for 3 (lane 3h) or 6 hours (lane 6h) or with 25 cycles (2 min 37°C+5 min 16°C, lane 25) or 50 cycles (lane 50), and without BsaI enzyme (lane nb). The arrow indicates the 1.17 kb linear assembled GFP gene product. (C) Ethidium bromide-stained gels of 72 minipreps digested with XmaI and AvrII (expected fragment sizes: 4.6 kb, 945 and 555 bp), obtained from restriction-ligations performed for 6 h 37°C (6 h), for 25 or 50 cycles (25×/50×), with normal ligase (nl) or high concentration ligase (hcl). Numbers indicate minipreps with an incorrect restriction pattern, and stars indicate constructs that consist of dimers (same restriction pattern as monomers). V, vector pX-lacZ.

    Journal: PLoS ONE

    Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

    doi: 10.1371/journal.pone.0005553

    Figure Lengend Snippet: Assembly of a GFP construct from 10 plasmids. (A) Construct maps. Input modules contain a core region C flanked by BsaI restriction sites in opposite orientation composed of a recognition site (B, ggtctcn, B , ngagacc) and a 4 nucleotide cleavage site (boxes flanking the core region). pX-LacZ, acceptor vector. pGFPi, resulting construct. Restriction sites for AvrII and XmaI are shown as white arrows. (B) Ethidium bromide-stained gel with products obtained by restriction-ligation of the 9 input module plasmids. M: GeneRuler 1kb DNA Ladder Plus from Fermentas. Restriction-ligation was performed at 37°C for 3 (lane 3h) or 6 hours (lane 6h) or with 25 cycles (2 min 37°C+5 min 16°C, lane 25) or 50 cycles (lane 50), and without BsaI enzyme (lane nb). The arrow indicates the 1.17 kb linear assembled GFP gene product. (C) Ethidium bromide-stained gels of 72 minipreps digested with XmaI and AvrII (expected fragment sizes: 4.6 kb, 945 and 555 bp), obtained from restriction-ligations performed for 6 h 37°C (6 h), for 25 or 50 cycles (25×/50×), with normal ligase (nl) or high concentration ligase (hcl). Numbers indicate minipreps with an incorrect restriction pattern, and stars indicate constructs that consist of dimers (same restriction pattern as monomers). V, vector pX-lacZ.

    Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

    Techniques: Construct, Plasmid Preparation, Staining, Ligation, Concentration Assay

    DNA shuffling strategy. (A) Two DNA ends terminated by the same 4 nucleotides (sequence f, composed of nucleotides 1234, complementary nucleotides noted in italics) flanked by a BsaI recognition sequence, B, form two complementary DNA overhangs after digestion with BsaI. (B) For shuffling, genes of interest are aligned, and recombination points consisting of 4 nucleotide sequences (f1 to fn+1) are defined on conserved sequences. Module fragments (core sequence, C1 to Cn, plus flanking 4 nucleotide sequences) are amplified by PCR and cloned in an intermediate cloning vector. Module fragment plasmids and the acceptor vector are assembled in one restriction-ligation with BsaI and ligase. S1 and S2, two different selectable markers. Z, lacZ alpha gene fragment.

    Journal: PLoS ONE

    Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

    doi: 10.1371/journal.pone.0005553

    Figure Lengend Snippet: DNA shuffling strategy. (A) Two DNA ends terminated by the same 4 nucleotides (sequence f, composed of nucleotides 1234, complementary nucleotides noted in italics) flanked by a BsaI recognition sequence, B, form two complementary DNA overhangs after digestion with BsaI. (B) For shuffling, genes of interest are aligned, and recombination points consisting of 4 nucleotide sequences (f1 to fn+1) are defined on conserved sequences. Module fragments (core sequence, C1 to Cn, plus flanking 4 nucleotide sequences) are amplified by PCR and cloned in an intermediate cloning vector. Module fragment plasmids and the acceptor vector are assembled in one restriction-ligation with BsaI and ligase. S1 and S2, two different selectable markers. Z, lacZ alpha gene fragment.

    Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Ligation

    Shuffling of trypsinogen. (A) Alignment of the aminoacid sequence of bovine cationic trypsinogen (BC), bovine anionic trypsinogen (BA) and human cationic trypsinogen (HC). Nucleotide sequence of the chosen recombination sites is shown. (B) Map of the 27 trypsinogen module plasmids, the acceptor vector, and of an example of one of the resulting shuffled construct obtained. B, BsaI restriction site. S, K: spectinomycin and kanamycin resistance genes. RB/LB, T-DNA right and left borders. AttB, Phage C31 recombination site, N tobamoviral 3′ non-translated region, T, Nos terminator. (C) Ethidium bromide-stained gels of 28 minipreps prepared from single colonies (1 to 24) or from 4 libraries (L1–4, approximately 700 clones in each) digested with XmaI (incorrect pattern 1, 2, 7 and 17).

    Journal: PLoS ONE

    Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

    doi: 10.1371/journal.pone.0005553

    Figure Lengend Snippet: Shuffling of trypsinogen. (A) Alignment of the aminoacid sequence of bovine cationic trypsinogen (BC), bovine anionic trypsinogen (BA) and human cationic trypsinogen (HC). Nucleotide sequence of the chosen recombination sites is shown. (B) Map of the 27 trypsinogen module plasmids, the acceptor vector, and of an example of one of the resulting shuffled construct obtained. B, BsaI restriction site. S, K: spectinomycin and kanamycin resistance genes. RB/LB, T-DNA right and left borders. AttB, Phage C31 recombination site, N tobamoviral 3′ non-translated region, T, Nos terminator. (C) Ethidium bromide-stained gels of 28 minipreps prepared from single colonies (1 to 24) or from 4 libraries (L1–4, approximately 700 clones in each) digested with XmaI (incorrect pattern 1, 2, 7 and 17).

    Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

    Techniques: Sequencing, Plasmid Preparation, Construct, Staining, Clone Assay

    Adaptation of GoldenBraid as a rapid, modular and robust system for novel applications in D. discoideum . ( A ) In a single test tube in a matter of hours, a GoldenBraid reaction can assemble a transcriptional unit into an α-level backbone from many (even > 7) individual DNA parts (top; P = promoter, CDS = coding DNA sequence, T = terminator). In a second, similar reaction, 2–5 α-level units can be combined into a single Ω-level vector backbone (bottom; U = transcriptional unit). Two Ω-level inserts comprising multiple transcriptional units can then be combined into an α-level backbone and so on, allowing iterative assembly until vector capacity becomes limiting (not shown). pUPD2 (red line) is the backbone that contains individual domesticated genetic elements (red boxes). The α-level backbones are denoted as black lines and the Ω-level backbones are denoted in green. Four-base sequences denote the grammar, lacZ denotes the E. coli beta-galactosidase gene, which is used in the blue-white screening. The striped arrows and the symbols they surround indicate the thermal cycles and the enzymes: ligase at 16°C in yellow, and BsaI and BsmBI at 37°C in magenta and purple, respectively. ( B ) Because of GoldenBraid's modularity, the present library of 99 GoldenBraid D. discoideum genetic elements (red bars) can be used to generate hundreds of transcriptional units (black and white bars, α1- and α2-levels, respectively), and hundreds of thousands of assemblies (green bar). The y-axis is in logarithmic scale and values are indicated inside each bar. ( C ) Cloning attempts (97) harboring properly assembled vectors are plotted such that each circle is an independent attempt, and the y-axis shows the fraction of desired clones (% of white colonies over total). In general, we miniprepped 2–4 clones corresponding to each GoldenBraid reaction. In all cases, at least one miniprepped clone was assembled correctly as determined by restriction enzyme fingerprinting. Colors match the above cloning level as indicated on the x-axis and the horizontal black lines indicate the average. ( D ) We adapted two new fluorescent proteins as indicated on the right of each panel, transformed them into D. discoideum cells and photographed the cells to illustrate proper expression and fluorescence. tagBFP scale bar = 10 μm; mIFP scale bar = 20 μm.

    Journal: Nucleic Acids Research

    Article Title: A GoldenBraid cloning system for synthetic biology in social amoebae

    doi: 10.1093/nar/gkaa185

    Figure Lengend Snippet: Adaptation of GoldenBraid as a rapid, modular and robust system for novel applications in D. discoideum . ( A ) In a single test tube in a matter of hours, a GoldenBraid reaction can assemble a transcriptional unit into an α-level backbone from many (even > 7) individual DNA parts (top; P = promoter, CDS = coding DNA sequence, T = terminator). In a second, similar reaction, 2–5 α-level units can be combined into a single Ω-level vector backbone (bottom; U = transcriptional unit). Two Ω-level inserts comprising multiple transcriptional units can then be combined into an α-level backbone and so on, allowing iterative assembly until vector capacity becomes limiting (not shown). pUPD2 (red line) is the backbone that contains individual domesticated genetic elements (red boxes). The α-level backbones are denoted as black lines and the Ω-level backbones are denoted in green. Four-base sequences denote the grammar, lacZ denotes the E. coli beta-galactosidase gene, which is used in the blue-white screening. The striped arrows and the symbols they surround indicate the thermal cycles and the enzymes: ligase at 16°C in yellow, and BsaI and BsmBI at 37°C in magenta and purple, respectively. ( B ) Because of GoldenBraid's modularity, the present library of 99 GoldenBraid D. discoideum genetic elements (red bars) can be used to generate hundreds of transcriptional units (black and white bars, α1- and α2-levels, respectively), and hundreds of thousands of assemblies (green bar). The y-axis is in logarithmic scale and values are indicated inside each bar. ( C ) Cloning attempts (97) harboring properly assembled vectors are plotted such that each circle is an independent attempt, and the y-axis shows the fraction of desired clones (% of white colonies over total). In general, we miniprepped 2–4 clones corresponding to each GoldenBraid reaction. In all cases, at least one miniprepped clone was assembled correctly as determined by restriction enzyme fingerprinting. Colors match the above cloning level as indicated on the x-axis and the horizontal black lines indicate the average. ( D ) We adapted two new fluorescent proteins as indicated on the right of each panel, transformed them into D. discoideum cells and photographed the cells to illustrate proper expression and fluorescence. tagBFP scale bar = 10 μm; mIFP scale bar = 20 μm.

    Article Snippet: For assembling initial transcriptional units into α-level vectors, we used 40 ng of the appropriate domestication-level parts, 75 ng of the desired α-level vector, and BsaI restriction enzyme (NEB).

    Techniques: Sequencing, Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Fluorescence

    Comparison of type II and IIS restriction enzyme-mediated protein tagging strategy ( a ) Type II restriction enzymes (TII-es) recognize palindromic DNA sequences. For example, EcoRI recognizes 5′-GAATTC-3′ (marked by top curly bracket) and creates 4 base pairs overhangs highlighted in red. ( b ) Single or double type II restriction enzymes cassette (highlighted in blue box) for traditional protein tagging. Note that in all destination clones, varying junction sequences exist adjacent to both sides of the tag. ( c ) Type IIS restriction enzymes (TIIS-es) recognize non-palindromic, asymmetrical DNA sequences. For example, BsaI recognizes 5′-GGTCTC-3′ (marked by top curly bracket) and cleaves DNA one bp away (indicated by the two arrows), producing N 2 ′N 3 ′N 4 ′N 5 ′ custom sticky end (highlighted in red). N indicates four bases of DNA, including A, T, G and C. Apostrophe (’) indicates the complementary base of the DNA. ( d ) Type IIS restriction enzyme DNA cassette (TIIS DNA cassette highlighted in blue box) for precision tagging. Note that on both ends of a tag, the flanking sequences (such as BsaI-released 5′-N 2 ′N 3 ′N 4 ′N 5 ′ and 5′-N 2 N 3 N 4 N 5 belong to gene-specific sequences including SP or gene of interest indicated by two closely dotted lines. After Tag replaces type IIS DNA cassette, a scarless tagging clone can be generated. Comparison between traditional and precision tagging were summarized in the bottom table. * Gibson assembly sometimes fails due to certain DNA sequences such as repetitive region or creating one or two nucleotides deletion.

    Journal: Biochemical and biophysical research communications

    Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning

    doi: 10.1016/j.bbrc.2017.05.153

    Figure Lengend Snippet: Comparison of type II and IIS restriction enzyme-mediated protein tagging strategy ( a ) Type II restriction enzymes (TII-es) recognize palindromic DNA sequences. For example, EcoRI recognizes 5′-GAATTC-3′ (marked by top curly bracket) and creates 4 base pairs overhangs highlighted in red. ( b ) Single or double type II restriction enzymes cassette (highlighted in blue box) for traditional protein tagging. Note that in all destination clones, varying junction sequences exist adjacent to both sides of the tag. ( c ) Type IIS restriction enzymes (TIIS-es) recognize non-palindromic, asymmetrical DNA sequences. For example, BsaI recognizes 5′-GGTCTC-3′ (marked by top curly bracket) and cleaves DNA one bp away (indicated by the two arrows), producing N 2 ′N 3 ′N 4 ′N 5 ′ custom sticky end (highlighted in red). N indicates four bases of DNA, including A, T, G and C. Apostrophe (’) indicates the complementary base of the DNA. ( d ) Type IIS restriction enzyme DNA cassette (TIIS DNA cassette highlighted in blue box) for precision tagging. Note that on both ends of a tag, the flanking sequences (such as BsaI-released 5′-N 2 ′N 3 ′N 4 ′N 5 ′ and 5′-N 2 N 3 N 4 N 5 belong to gene-specific sequences including SP or gene of interest indicated by two closely dotted lines. After Tag replaces type IIS DNA cassette, a scarless tagging clone can be generated. Comparison between traditional and precision tagging were summarized in the bottom table. * Gibson assembly sometimes fails due to certain DNA sequences such as repetitive region or creating one or two nucleotides deletion.

    Article Snippet: All the enzymes including type II restriction enzymes (EcoRI, BamHI, SalI and Bau36I), type IIS restriction enzymes (BsaI, BbsI and BsmBI), T4 DNA ligase, Phusion enzyme, and T5 exonuclease were purchased from New England BioLabs.

    Techniques: Clone Assay, Generated

    Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.

    Journal: bioRxiv

    Article Title: High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

    doi: 10.1101/2020.05.12.091991

    Figure Lengend Snippet: Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.

    Article Snippet: For controls, target plasmids and empty pUC19 were linearized by restriction enzyme digestion using BsaI-HF and nicked using a nicking enzyme Nt.BspQI (New England Biolabs).

    Techniques: Plasmid Preparation, Incubation

    High-throughput in vitro analysis of Cas9 mismatch tolerance. (A) Outline and workflow of the high-throughput in vitro cleavage assay. (B) Representative agarose gel showing time course cleavage of negatively supercoiled (nSC) plasmid containing a fully matched PS4 target (pTarget PS4, left) and plasmid library PS4 (pLibrary PS4, right) by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); (-r) = pTarget or pLibrary incubated with Cas9 only at 37 °C for the longest time point in the assay (3 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (C) Overall cleavage of the pLibrary PS4 by Cas9 indicating the decrease in supercoiled (nSC) pool and appearance of nicked (n) and linear (li) pools over time. The 0 time point is the quantification of the negative control pLibrary (i.e. pLibrary run on a gel after preparation as represented in Fig. S2A). Values plotted represent an average of two replicates. Error bars are SEM.

    Journal: bioRxiv

    Article Title: High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

    doi: 10.1101/2020.05.12.091991

    Figure Lengend Snippet: High-throughput in vitro analysis of Cas9 mismatch tolerance. (A) Outline and workflow of the high-throughput in vitro cleavage assay. (B) Representative agarose gel showing time course cleavage of negatively supercoiled (nSC) plasmid containing a fully matched PS4 target (pTarget PS4, left) and plasmid library PS4 (pLibrary PS4, right) by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); (-r) = pTarget or pLibrary incubated with Cas9 only at 37 °C for the longest time point in the assay (3 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (C) Overall cleavage of the pLibrary PS4 by Cas9 indicating the decrease in supercoiled (nSC) pool and appearance of nicked (n) and linear (li) pools over time. The 0 time point is the quantification of the negative control pLibrary (i.e. pLibrary run on a gel after preparation as represented in Fig. S2A). Values plotted represent an average of two replicates. Error bars are SEM.

    Article Snippet: For controls, target plasmids and empty pUC19 were linearized by restriction enzyme digestion using BsaI-HF and nicked using a nicking enzyme Nt.BspQI (New England Biolabs).

    Techniques: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control