Protocol Online logo
Top : Forum Archives: : Molecular Biology

Question about ligation! - Ligation of 6kb insert in pGEM T-vector (Dec/17/2007 )

Hi all,

I am trying to ligate a 6kb insert into a pGEM T-Vector (3kb). Since i need to use a proofreading polymerase, i first amplified the insert using KOD polymerase, then trying an ''A'' tailing protocol. However I wasn't successful. I then discovered Platinum Taq Hifi (from Invitrogen), an enzyme mixture composed of recombinant Taq DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum Taq Antibody which will able to me have the ''A'' overhang. But before trying ligate the product, I would like to get some feedback about ligating a large insert into a pGEM T-vector. Has anyone been successful? Any tips or suggestion? What insert:vector ratio would give me the most transformation efficiency (in DH5alpha)?

Thanks for everything in advance and happy holidays to everyone


instead of using (for example) a 3:1 insert to vector ratio, use a 1:3 insert to vector ratio. this is because the vector is small in comparison to the insert.