What is necessary to clone PCR product into a pGEM®-T vector?
All that is necessary for ligating a PCR product into a T-cloning vector is a single deoxyadenosine overhang at each of the 3′-ends. Some thermostable DNA polymerases, including Taq DNA polymerase, add a single deoxyadenosine onto the 3′-ends of PCR products in a template-independent fashion, making the PCR products ready for cloning. If you use an enzyme for PCR that produces a blunt-ended product, like a proofreading thermostable DNA polymerase, clean up the reaction to remove the enzyme and then treat the sample with 5 units Taq DNA polymerase (in 1X reaction buffer with 200µM dATP) for 15–30 minutes at 70°C. This reaction will add the required deoxyadenosine overhangs at each of the 3′-ends of the PCR product so it is then ready to be ligated into a T-cloning vector like the pGEM®-T Vector (Cat.# A3600, A3610) or the pGEM®-T Easy Vector (Cat.# A1360, A1380). More T Vector FAQs…
