Molecular Engineering Library

PEF has extensive knowledge in molecular engineering techniques, which include molecular cloning, site-directed mutagenesis (SDM), vector engineering and plasmid isolation. Find out how each technique may accelerate your research.

General information
Related articles

Molecular cloning

Molecular cloning is the first laboratory step in producing a recombinant protein. The aim of the process is to obtain an expression vector (also referred to as a plasmid) that carries the gene of interest (GOI).

The general workflow is described in the diagram below:

Frequently Asked Questions – Molecular Cloning

How do I select my cloning strategy?

There is no right or wrong cloning strategy. The decision depends on what resources you have available in your lab and their individual merits e.g. how fast is the strategy, how efficient is it etc. Useful articles on several cloning strategies can be found in related articles.

Do I have to codon optimise my gene?

Codon optimisation involves modifying the codons within a gene sequence to match the codon frequency of the intended expression host. It can theoretically enable higher protein production rates, however it is not critical for achieving successful protein expression.

Site-directed mutagenesis (SDM)

Vector engineering

Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.
Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. & Peleg, Y. Journal of structural biology 172, 34-44 (2010).

Circular polymerase extension cloning of complex gene libraries and pathways.
Quan, J. & Tian, J. PloS one 4, e6441 (2009).

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications
Berrow, N.S. et al. Nucleic acids research 35, e45 (2007).

Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.
Li, M.Z. & Elledge, S.J. Nat Meth 4, 251-256 (2007).

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.
Cabrita, L.D., Dai, W. & Bottomley, S.P. BMC biotechnology 6, 12 (2006).