DNA Manipulation About

DNA cloning and sequence manipulation are essential tools in all life science disciplines. 

In recent years, the SPU is using two Ligation Independent Cloning (LIC) strategies; Restriction Free (RF) and  Transfer-PCR (TPCR), based on whole plasmid amplification of the vector and the insert.

The RF (van den Ent and Lowe, Unger et al., Peleg and Unger) and TPCR (Erijman et al., Erijman, Shifman and Peleg) procedures are universal cloning methods allowing a precise insertion of a DNA fragment into any desired position within a circular plasmid. Both methods are based on synthesis of mega primers (up to several kbs) containing flanking target-vector specific sequences. The RF/TPCR methodologies were used successfully in a variety of applications to manipulate the target DNA sequence. These include cloning of multiple DNA fragments, insertion and/or deletion of specific DNA sequences, and single and multiple-site mutagenesis.

Unit activities

  • Engineering plasmid DNA, including:
    • DNA cloning
    • DNA mutagenesis (single and multiple alterations, deletion, insertion, etc...)
  • Consultation