Molecular cloning

A range of molecular cloning services are available at PEF, including:

Designing an effective cloning strategy

PEF will design and implement the optimal cloning strategy for your protein of interest.

DNA template generation

Includes: Gene assembly, Gene synthesis and Codon optimisation (optional)

Vector re-construction

Creating a vector field from experimental or computer generated data, usually with the goal of finding a differential equation model of the system.

Customised or high throughput molecular cloning

PEF offers a number of high throughput services in molecular cloning, expression, purification and characterisation.

Sequence verification

PEF can assist in confirming the DNA sequence of your gene.

Site-directed mutagenesis

SDM is a molecular technique used to introduce specific mutations (deletions/insertions/point mutation) to the GOI or vector sequence.

For all molecular cloning services, please contact us.
To view our list of available vectors, please visit our UQ Resource Centre.

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

A general methodology is described briefly below:

  1. Obtain/generate a DNA template of the GOI
  2. Ligate the GOI into an appropriate expression vector (ligation dependent cloning)
  3. Transform the expression vector into a bacterial strain
  4. Analyse bacterial clones to confirm integration of the GOI
  5. Sequence verification

Cloning Strategies

There are a variety of cloning strategies available and they can be broadly classified into 2 categories as shown in the diagram below.

Cloning Strategy Diagram

Useful articles on several of these cloning strategies can be found on the related articles page.

Site-directed Mutagenesis (SDM)

SDM is a molecular technique used to introduce specific mutations (deletions/insertions/point mutation) to the GOI or vector sequence. SDM is commonly used for mutations involving:

  • Restriction endonuclease recognition sites
  • Start or stop codons
  • Fusion tags and signal peptides
  • Optimisation of transcriptional elements on the vector

Codon Optimisation

Codon optimisation is a tool that can be used to improve expression levels of heterologous recombinant proteins. The process involves modifying codons within a gene sequence to match the codon frequency of the intended expression host, theoretically enabling higher production rates.

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).

PEF offers upskilling for staff and students in all aspects of recombinant protein production. If you would like to enquire about training in any of the services offered by PEF please contact us.

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