A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement
نویسندگان
چکیده
منابع مشابه
A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement
BACKGROUND In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. RESULTS In our case study, t...
متن کاملRapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5'-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatmen...
متن کاملRestriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.
In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactio...
متن کاملCloning-free PCR-based allele replacement methods.
Efficient homologous recombination permits the directed introduction of specific mutations into the yeast genome. Here we describe a cloning-free, PCR-based allele replacement method that simplifies allele transfer between yeast strains. The desired allele from one strain is amplified by PCR, along with a selectable/counterselectable marker. After transformation, the resident allele in the targ...
متن کاملA high-throughput cellulase screening system based on droplet microfluidics.
A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% pur...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: Microbial Cell Factories
سال: 2014
ISSN: 1475-2859
DOI: 10.1186/1475-2859-13-38