Simplified Method for Ligase-Free Cloning of PCR Products
نویسندگان
چکیده
منابع مشابه
Simplified method for ligase-free cloning of PCR products.
Cloning of polymerase chain reaction (PCR) products into plasmid vectors by conventional methods, relying either on recognition sequences built into the primers or taking advantage of the template-independent terminal transferase activity of Taq DNA polymerase to add an extra adenine to the 3′ end of the product, are often difficult (1,10,12). In recent years, numerous cloning strategies (10,13...
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 کاملFastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method
BACKGROUND Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. RESULTS Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR a...
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 کاملDirectional cloning of PCR products.
INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to re...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1996
ISSN: 0736-6205,1940-9818
DOI: 10.2144/96215bm17