DNA cloning and sequence manipulation are fundemental techniques across all life science disciplines. Cloning a gene or any DNA sequence into a plasmid vector or introducing specific mutations can be achievied through various methods, including traditional ligation dependent cloning (LDC), which relies on restriction enzymes followed by ligation, or ligation independent cloning (LIC).
At the Structural Proteomics Unit (SPU) we primerly employ two LIC-based strategies; Restriction-Free (RF) and Transfer-PCR (TPCR). Both approaches involve whole plasmid amplification of the vector and the insert. The RF method is a two steps process coomenly used for cloning, wheras TPCR is a single step method primerely used for mutagenesis.
The RF and TPCR methods enable precise insertion of a DNA fragments at any desired position within a circular plasmid. Both rely on the generation of megaprimers (up to several kbs) that contain flanking target-vector specific sequences. The RF/TPCR methodologies have been successfully applied to a range of DNA manipulations, including cloning of multiple DNA fragments, insertion and/or deletion of specific DNA sequences, and single or multiple-site mutagenesis.
Unit activities
- Engineering plasmid DNA, including:
- DNA cloning by different methods
- Restriction enymes based cloning
- Restriction Free (RF) cloning
- Transfer PCR (TPCR)
- Gibson assembley
- In-Fusion cloning
- DNA mutagenesis by different methods
- Quik-Change mutagenesis
- Transfer-PCR (TPCR)
- Inverse-PCR
- Single and multiple site-directed mutagenesis
- DNA cloning by different methods
- Consultation
- Primer design for cloning, mutagenesis and sequencing
- Plasmid design for protein expression and cell biology
- Consulation on DNA manipulation strategies