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

      1 ,
      Journal of biochemical and biophysical methods
      Elsevier BV

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          Abstract

          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.

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          Author and article information

          Journal
          J Biochem Biophys Methods
          Journal of biochemical and biophysical methods
          Elsevier BV
          0165-022X
          0165-022X
          Apr 30 2006
          : 67
          : 1
          Affiliations
          [1 ] MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. fent@mrc-lmb.cam.ac.uk
          Article
          S0165-022X(06)00002-9
          10.1016/j.jbbm.2005.12.008
          16480772
          0fc00d47-7d14-49be-992a-3b657d3b49ea
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