Can't clone a 5 kb insert

[gw forsyth]

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?

[kevin_a]

Have you considered that some of the bits you want to clone could be toxic to the bacteria? See if you can clone the smaller pieces and then drop in the other inserts in a later step.

It sounds odd, but I had a similar problem trying to clone conserved promoter regions from 12 different animal species. Some would clone and some would not even after going through the trouble of engineering restriction sites into ends of the sequences to avoid blunt end cloning.

[gw forsyth]

The three smaller pieces cloned into pcDNA2.1 on the first try. The whole thing could be toxic but I don't expect it to be expressed in Top 10s???

Incidentally, I lied about no inserts. We see "ghost bands" of the right size on pcr screening. We call them ghost bands because:
1) they are much lighter than positive controls
2) when PEG prepped they give garbage sequence
3) no-template controls are clean
4) streaking them out to reclone isolated colonies does not help

Is there any kind of super repressor transformation host that will prevent a toxic response to the protein? We have to get it into a vector to do some gene therapy trials.

[admin]

Hello Gw,

I would try sub cloning first into a TOPO4 sequencing vector if you can PCR the 5kb fragment maybe with long PCR with fidelity (Gold). The only problem is you would have to do multiple sequencing to check if the 5kb is ok.
TOPO4 is a great vector for subcloning if you can pcr your fragment, it has even a death domain and blue/white selection. Once you sub clone its quite easy to clone it out as the TOPO4 MCS has tons of restriction enzyme sites.