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