RF cloning: a restriction-free method for inserting target genes into plasmids

J Biochem Biophys Methods. 2006 Apr 30;67(1):67-74. doi: 10.1016/j.jbbm.2005.12.008. Epub 2006 Feb 3.


Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics.

MeSH terms

  • Cell Line
  • Cloning, Molecular / methods*
  • DNA Primers
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genetic Vectors
  • Humans
  • Plasmids*
  • Polymerase Chain Reaction


  • DNA Primers