Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 40 article reviews
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1) Product Images from "Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging"
Article Title: Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging
Journal: Nucleic Acids Research
Figure Legend Snippet: Fabrication of torsionally-constrained DNA with length up to 10 kb by golden gate assembly. ( A ) 0.9 kbp DNA handles were made by PCR using either biotin or digoxigenin dUTP incorporation. Unlabelled PCR sections of 2.1 kb with encoded BsaI sites were separately made. The designed structure after joining of the purification PCR parts by golden gate assembly is 10.1 kb in length. ( B ) Agarose gel image of the product from the golden gate assembly reaction. The band at ∼10 kb reacts with both streptavidin and anti-digoxigenin indicating successful dual labelling. ( C ) Schematic of magnetic tweezers used for force spectroscopy measurements. ( D ) Example torsional response of single DNA tether showing characteristic supercoiling at low force. ( E ) Force extension curves of six DNA tethers in different colours. ( F ) Schematic of flow stretching of DNA molecules. ( G ) TIRF image of multiple beads stretched by a flow of 0.5 μl/s.
Techniques Used: Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Spectroscopy
Figure Legend Snippet: Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.
Techniques Used: Polymerase Chain Reaction, Synthesized, Incubation
Figure Legend Snippet: Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.
Techniques Used: Construct, Polymerase Chain Reaction, Incubation, Agarose Gel Electrophoresis
Figure Legend Snippet: Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.
Techniques Used: Polymerase Cycling Assembly, Generated, Sequencing, Amplification, Polymerase Chain Reaction, Construct, Incubation
2) Product Images from "Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector). Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector"
Article Title: Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector). Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector
Journal: Biotechnology and Bioengineering
Figure Legend Snippet: Schematic representation of the MIDAS‐P assembly system for plant expression. The system consists of two entry vectors, pWHITE and pBLUE, for cloning genes of interest and alternate sub‐cloning in the binary destination (expression) vector pMIDAS. The first transcriptional unit is constructed in pWHITE and transferred into pMIDAS using the type IIS restriction enzyme BsaI. A second transcriptional unit in pBLUE can subsequently be transferred into pMIDAS using BsmBI. Further TUs can be added by alternating transfer from pWHITE and pBLUE. The inclusion of lacZα in pMIDAS and pBLUE allows blue/white screening at each stage. The destination vector pMIDAS also has right and left T‐DNA borders for Agrobacterium ‐mediated plant transformation. GOI, gene of interest; P, promoter; pA, terminator and polyA signals; SAR, scaffold attachment region; UTR, untranslated region
Techniques Used: Expressing, Clone Assay, Subcloning, Plasmid Preparation, Construct, Transformation Assay