Insert restriction enzyme cutting-free cloning strategy for expression plasmid construction
نویسندگان
چکیده
منابع مشابه
Restriction digestion monitors facilitate plasmid construction and PCR cloning.
Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indi...
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 site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris
BACKGROUND Tagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector g...
متن کاملEnzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites.
We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation-hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This 'enzyme-free cloning'...
متن کاملDNA cloning without restriction enzyme and ligase.
One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form h...
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ژورنال
عنوان ژورنال: Biotechnology & Biotechnological Equipment
سال: 2017
ISSN: 1310-2818,1314-3530
DOI: 10.1080/13102818.2017.1351310