Molecular Biology

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    Protocols in Current Issue
    Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae
    Authors:  Tokiaki Takemura, Sousuke Imamura, Yuki Kobayashi and Kan Tanaka, date: 04/05/2019, view: 477, Q&A: 0
    [Abstract] The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same ...
    Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana
    Authors:  Teruhiko Kuroyanagi, Maurizio Camagna and Daigo Takemoto, date: 08/05/2018, view: 1310, Q&A: 0
    [Abstract] Plant species produce a wide variety of antimicrobial metabolites to protect themselves against potential pathogens in natural environments. Phytoalexins are low molecular weight compounds produced by plants in response to attempted attacks of pathogens. Accumulation of phytoalexins in attacked plant tissues can inhibit the growth of penetrating ...
    Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases
    Authors:  Zhen Xu, Yan-Ning Rui, John P. Hagan and Dong H. Kim, date: 02/05/2018, view: 2937, Q&A: 0
    [Abstract] Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, ...
    Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
    [Abstract] This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible ...
    Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
    Authors:  Rachael M. Giersch and Gregory C. Finnigan, date: 09/20/2017, view: 5454, Q&A: 0
    [Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded ...
    Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants
    Authors:  Xu Tang, Zhaohui Zhong, Xuelian Zheng and Yong Zhang, date: 09/05/2017, view: 5783, Q&A: 0
    [Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, ...
    Using CRISPR-ERA Webserver for sgRNA Design
    Authors:  Honglei Liu, Xiaowo Wang and Lei S. Qi, date: 09/05/2017, view: 5297, Q&A: 0
    [Abstract] The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/ ...
    Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
    Authors:  Xiaoou Jiang and Volker Patzel, date: 08/05/2017, view: 3913, Q&A: 0
    [Abstract] Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA ...
    Dense sgRNA Library Construction Using a Molecular Chipper Approach
    Authors:  Jijun Cheng, Wen Pan and Jun Lu, date: 06/20/2017, view: 4741, Q&A: 0
    [Abstract] Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for ...
    Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression
    Authors:  Xiaoou Jiang and Volker Patzel, date: 06/05/2017, view: 4188, Q&A: 0
    [Abstract] A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo ...



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