We are trying to ligate three smaller cDNA frags together using unique restriction sites and then clone 5 kb gel purified ligation product into pcDNA 3.1 TA V5 his TOPO. After ligation both ends are blunt with Eco RV, so we put the gel purified 5 kb ligation product in thermocycler for 10 min with Taq and dATP to get A overhangs. Using 50 ng of this insert, and 30 min to an hour to get TOPO insertion into vector we can't get any positive clones by either electroporation into DH5alphas or using Invitrogen's chem competent Top10s. Last try had 10 Amp resistant Top 10 colonies. None have any inserts by pcr screening.
We are banging our heads on the wall. Any ideas?