Viral Vector cloning frustration!

[Lyanesse]

Hello all,

I am trying to clone a 1.5kB gene into a lentiviral vector (has ampR gene) that we use routinely in the lab. I've troubleshooted through a few problems over the last two months, but now I'm stumped and I need help! I'm relatively new at this, but even the molecular bio guy in our lab who does this all the time doesn't know what to do. Here's what I have done so far:

PCR gene with primers adding BamHI restriction sites to each end
obtained PCR product of correct size
cloned this into TOPO successfully (I got colonies) using TOP10 chemically competent cells, selected 4 colonies
digested vector and plasmid with BamHI overnight at 37C
dephosphorylated vector with CIP for 1h at 37C, cleaned with PCR clean kit (Qiagen)
ran plasmid digest on gel, obtained fragment of correct size in all but one digest
purified gel fragments using Promega wizard extraction kit
set up ligation reaction with 1:0 and 2:1 ratios of vector:insert (approximately 150ng total DNA in a 10uL ligation reaction volume)
ligated overnight at 16C
used 5uL to transform ~30uL TOP10 competent cells
incubate in 1mL SOC for 45min, then plate 250uL on LB-Amp plates
the next day I got 5-10 colonies on each 2:1 plate and very few or no colonies on the 1:0 plates

From those plates, I selected 12 colonies (4 from each plate), grew those up in LB-Amp for 6h and digested the DNA overnight at 37C with BamHI to check for the plasmid of the correct size. Of the 12, I only got one that had an insert of the correct size, and sent it for sequencing, hoping it would be in the proper orientation since I was using a non-directional strategy.

Now here's the kicker--the sequencing showed that the insert appeared to be of the correct orientation, however the gene was truncated at about 430 bp, and the rest of the DNA was junk! It wasn't from the vector, and there is no Bam restriction site where it was truncated...

Could this be from plasmid recombination or what? And based on the fact that I only got 1/12 colonies with any part of my insert, I think something else must be wrong...

Any suggestions would be greatly appreciated!! Thank you in advance for your help.

[Andriy]

Lyanesse,
BamHI has a very high star activity. Cutting for longer than 45 min leads to chewing up of the fragment ends. After overnight digestion you could have no sticky ends left on the DNA.
My advice - BamHI cutting in NEBuffer 3 for 30 min.
Let me know how it goes!