Protein Expression and Purification Core Facility

PEPCF expresses proteins in bacteria, insect and mammalian cells and uses a variety of chromatographic and biophysical techniques for protein purification and characterization.

TA and TOPO®-TA cloning

The TA cloning method takes advantage of the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3′-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3′-T overhangs. DNA polymerases with proofreading activity, such as Pfu polymerase, cannot be used because they provide blunt-ended PCR products.

For example, the pTA plus vector (based on the pPCR-Script Amp from Stratagene) can be used for TA cloning. Digestion of this vector in two sequential reactions with BamHI and XcmI results in a linearized vector with 3′-T overhangs and a low background of non-recombinants.

TA cloning is often combined with Topo® cloning, which takes advantage of the dual function of the enzyme DNA topo-isomerase I, which can act both as a restriction enzyme and a ligase. TOPO-TA vectors are usually already linearized and activated with topo-isomerase I, which allows direct ligation of PCR products with a 3’-A overhang in 5 minutes. TOPO-TA vectors are available from Thermo Fischer Scientific.