Ligating with large insert - (Sep/13/2007 )
I am on the final stage of making my construct and I need to ligate a 11kbp insert into a 10.5kbp vector. I have tried this once so far with no luck. Are there any tricks to help ligations of this sort where the insert and the vector are roughly the same size and pretty big?
At the moment I am digesting both with XhoI and NotI, dephosphorylating the vector with SAP, gel purifying and using T4 ligase at 16 C before transforming into TOP10 cells. I used a variety of ratios of vector:insert.
unfortunately i don't believe there are any special trick for large fragments. I think it is more or less a test on how well you have mastered the back art of ligation. The steps for ligating 1kb inserts is about the same with ligating a 190kb insert (aside from electrolution to free the DNA from the gel matrix after purifying the insert.)
Do not over dephosphorylate.
Use high concentrations of your insert and vector. (1:1 ratio)
Can you conduct a kill digest? Do you have a unique restriction site between the XhoI and NotI site?
You can try ligating at 4 Celsius overnight. It sometimes helps.
Thanks for advice.
At the moment I add 1ul SAP to my restriction digests and incubate at 37 C for 30 minutes, inactivate at 65 C for 20 minutes. Is this about right?
sound like too much. How much DNA are you dephosphorylating?
What exactly do you mean with "no luck"? No transformants, or a lot of them but empty vector?
If no transformants at all: restriction of vector was fine but most likely dephos ruined it for you (or too much UV-exposure during purification maybe?), if a lot of them but empty the cutting part didn't go well.
If dephos is the key: use AAP from NEB (was way more gentle in my hands that the others).
(I do take for granted your competent cells are fine, talking of which: are you electroporating or heath-shocking?).
I'm with the concensus. I don't see why you have to SAP treat your vector. If it is cut well, there should be little background. UV exposure during gel imaging can be a major problem, especially with long fragments. Where does the insert come from? Is it a PCR fragment? If so, does it have sufficient 5' overhang? I would be trying electroporation for a plasmid this size. You should not have to do anything beyond a 30 minute room temperature ligation for this to work.