3fragment ligation- troubleshooting - Cloning (Sep/03/2010 )

Hello All

I have cloned tanc1(approximately 5.6kb in size) into pbuescript vector(3kb approx.) successfully. Tanc1 was big enough and so I looked at the restriction sites and cut it into two fragments- one 2kb A fragment and another 4 kb B fragment. The 2kb fragment had Sal1 and Hind3 ends and the 4kb fragment has Xho, Hind3 ends so that A and B can ligate together through the Hind3 ends. I got the A insert and B insert successfully in pbluescript and I got the sequences to confirm its tanc1. Now I need to get the tanc1 out of pBS and clone it into the expression vector pCMV(approx. 4kb).
I used teh following strategy:
I cut pBS+A clone with Sal,Hind3 to pop out the insert A.
I cut pBS+B clone with Xho,Hind3 to pop out the insert B.
I looked at the pCMV tag1 vector map and selected enzymes Sal1, Xho1 in its MCS for cloning.
This is how my ligation looks :
pCMV cut with Sal and Xho
Insert A
Insert B
Ligase
T4 DNA buffer
ddh2O

I have tried this strategy thrice now and I am not getting any results.
Can someone help me with this?
Thank you in advance.

My suggestion is to abandon this current strategy. As a multiway ligation strategy, it is rather difficult as the ends of the DNA fragments are not incompatible with each other. This is further complicated as the ends of the vector are compatible, which means that there is a high probability of recovering self ligated vector. (ie Xho and SalI produce the same overhangs)

I would suggest that the ligation strategy be modified. A third restriction enzyme (more accurately a third cohesive sequence) be used. As an example, BamHI

THus we have

BamHI-Fragment A-HindIII
HindIII-Fragment B- XhoI
BamHI-vector-XhoI

I would also recommend that SalI not to be used. Ligations with SalI are difficult, it is believed that the SalI enzyme modifies the ends of the DNA molecule it cuts.. making ligation difficult. We observe that the DNA is cut but it doesn't ligate.

I would also strongly recommend that you avoid using quick ligation or ligation at room temperature. Multiway ligation work best using normal T4 DNA ligase and incubated at around 16 C.

Obviously, I'm missing something here...

If you have "cloned tanc1 into pbuescript successfully" as a single insert, and now want tanc1 in pCMV, why are you doing a three-way ligation? Why not just recover the whole 5.6 kb tanc1 insert from pBluescript and clone it into pCMV directly?