A variety of methods have been developed for cloning PCR products, including blunt-end cloning (1), restriction cut back (2), ligation-independent cloning (3), uracil DNA–glycosylase (UDG) treatment of uracil-containing deoxyoligonucleotide primers (4,5) and TA cloning (6–8). Blunt-end cloning of PCR products often requires treatment of PCR products to polish the ends (9). Even with treatments,...

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...

D) DIGESTION WITH Hind III AND EcoRI PROTOCOL, a) cDNA synthesis by Reverse Transcriptase is initiated with oligo(dT) according to Gubler and Hofmann (1) . b) The double stranded cDNA is methylated, and (c) a synthetic CCTTGAATTCAAGCNH oligonucleotlde pGCTTGAATTCAAGC CCAACTTAACTTCCNN is added. This generates a Hindlll recognition sequence (AAGCTT) when ligated to the oligo (dA) track at the 3 t...

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) ve...

We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfull...

Background: Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand. Objectives: For increasing the ethyl acetate yield of industrial brewer’s yeast strain, all the ATF1 alleles were overexpressed t...

Recombinant gene strategies using fusion tags for purification are essential procedures to obtain large protein quantities. However, most cloning systems result in recombinant proteins with added amino acids inexistent in their native forms which can lead to significant changes in protein properties. An original and simple cloning strategy is proposed to obtain proteins identical in amino acid ...

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vect...