Help: incompatible RE site subcloning - (Dec/15/2009 )
Vector A: 5' NheI, insert, 3' AflII (no other RE sites)
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!
As far as I know, 3 way ligation could be very complicated. The ratios, DNA quality, proper digests, phosphatase treatment.. too many difficult parameters to control. If I were you I'd serioulsy consider the possibility of PCRing the insert from vector A with a reverse primer having BsiW1 site (see below)
If this is the end of your DNA to be cloned from vector A, 5'.....AGTGAGATCGGAACCTG3'AflII site
design the reverse primer like this: 5'BsiW1 siteCAGGTTCCGATCT....3'