TA cloning and 3 fragment ligation problems - (Jul/27/2008 )

Help - I have to link three genes together in an expression vector, two are already linked in a TA vector and the third is in the desired expression vector. I attempted double digest and ligation to the expression vector which failed. Problem is that the expression vector construct and the TA have a XhoI site both before and after the desired constructs. I need to ligate gene 2 and gene 3 by use of a unique restriction site, but the XhoI sites cause three fragments to ligate:

Gene 1+2, gene 3, and the XhoI cut expression vector (cut on both ends).

As I said attempts at three fragment ligation have failed and I opted to attempt to move the gene3 into the TA with gene 1+ 2. The plan was to ligate gene 3 into the TA then restrict with XhoI (both ends) and ligate to cut expression vector. Then I hit another roadblock and realized the PCR to amplify gene 1 had incorporated the same set of restriction sites 5' of my gene that lie 3' in the TA vector (gene was amplified out of a pcDNA3.1 construct), so instead of being able to linearize the TA construct and ligate my gene 3, I keep cutting gene 1+2 out.

I attempted several times to cut my gene 1+2 out of TA using EcoRI, fill-in 3' recesses and add A's, then religate to TA (this would remove the set of restriction sites 5' of my genes). However, these ligation attempts also keep failing. Have tried Taq + dNTP and Klenow followed by Taq to fill in and add A's. Positive ligation controls and transformation controls work well. Can anyone help? I am going crazy with this project.

Personally I would simply PCR amplify each insert using primers carrying the unique restriction site that you desire. You can then design an simple ligation strategy that will get your inserts into the expression vector. Primers are relatively cheap to buy and proof reading DNA polymerase are relatively reliable and simple to use.