Bryksin and Matsumura’s 2010 article clearly details a simple method for creating recombinant plasmids using complementary overlapping ends, and remains one of the most popular articles on our site.
Lazinski and Camilli describe the addition of homopolymer tails of defined lengths to 3’ termini of genomic templates for use in subsequent PCR and cloning applications. The true value of this approach lies in the ability to use very low amounts of starting material to construct next-generation sequencing libraries.
In recent years, eliminating ligase from cloning (enzyme-free cloning) has clearly been a trend for developers. In this article, Yang et al. develop a technique to clone DNA using nicking DNA endonuclease (NiDE). Here, NiDe cassettes are created by flanking an insert with NiDE substrate sites, which can result in the generation of 14 base pair termini and enable subsequent cloning of a target sequence into a NiDE generated vector with high efficiency.
For techniques such as bimolecular fluorescence complementation (BiFC), the
expression of multiple genes is critical to success. Often times,
those genes are expressed from different vectors which can result in
unequal expression patterns that complicate BiFC analysis. In 2010,
Grefen and Blatt described a cloning system they developed called
‘2-in-1’ where two candidate genes can be cloned into an expression
vector simultaneously. By using a single vector for multi-gene
expression, the unequal gene expression concerns can be reduced for
researchers, lessening the need for complicated controls and
Two-hybrid screens have become an important tool when it comes to studying protein interactions. The examination of whole genomes is even possible now as complete clone sets exist for many species. In this article, Rajasekhar et al. describe the development of a new set of Gateway-suitable cloning vectors that can make the process of transferring large clone sets between vectors simpler and faster.
In this 2012 Benchmark article, Berlec and Strukelj describe an alternative approach to creating TA cloning expression plasmids for Lactococcus lactis based on the nisin-controlled expression system (NICE) plasmid pNZ8148. This TA cloning vector strategy is not exclusive to L. lactis and adds to the toolbox of methods for cloning PCR generated DNA fragments.
In vivo recombinational cloning in yeast has proven to be an efficient cloning methodology. However, the method has been limited in the past to using yeast vectors since most animal, insect, and bacterial vectors lack the required yeast origin of replication. In this article from 2006, Iizasa and Nagano detail the development of a new system capable of utilizing yeast-based in vivo vectors lacking the yeast replication origins for cloning in yeast.
Here, Matsumoto and Itoh perform a systematic analysis of the requirements for efficient ligation-free cloning. Focusing on overhang length and GC content, the authors detail how to design optimal overhangs for your ligation-free cloning experiments.
The ability to generate starting material for cloning or other experiments from limited amounts of sample can be a challenge. Rolling circle amplification is one approach to solving this problem but false-priming and primer-dimers can limit success of this technique. In this article, Takahashi et al. suggest using random RNA primers instead of DNA primers in the rolling circle amplification reaction. The authors' data demonstrated the ability to copy DNA more than 1012 fold using the approach – generating microgram amounts of amplification products.
In this article, author John Geiser details the construction of a series of galactose-inducible centromere-based yeast expression vectors that are able to quickly exchange cloned genes between vectors using Gateway® technology.
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