Vector B: 5' NheI, 3' BsiW1 (no other RE sites)
I am trying to subclone the insert in A into B without destroying BsiW1 because BsiW1 will be used later. NheI can be used at 5', but AflII and BsiW1 are not compatible. I don;t want to do PCR because the insert is kind of big.
I am thinking of using 2 oligos to make an adapter which will have AflII overhang and BsiW1 overhang, then do 3-way ligation. but don't the details: e.g. how to design the oligos, annealing conditions....
Is the 3-way ligation a good idea? Any other ideas to do this subcloning? You help is greatly appreciated!
Edited by Eric_W, 15 December 2009 - 11:36 AM.