Services

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

  • Designing an effective cloning strategy
  • DNA template generation
    • Gene assembly
    • Gene synthesis
    • Codon optimisation (optional)
  • Vector re-construction
  • Customised or high throughput molecular cloning
  • Sequence verification
  • Site-directed mutagenesis

For all molecular cloning services, please contact us. To view our list of available vectors, please visit our resource centre.

Training

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

For an overview of the services provided by PEF, please visit the PEF production pipeline.

General Information - Molecular Cloning

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.

Related Articles on Molecular Cloning

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

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

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

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

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