A few days ago I did a 3-piece ligation that didn’t turn out so well. Today’s ligation is more like the traditional ligation that I was supposed to do with the exception that I’m using the pALS anchor instead of the pRL anchor.
I mentioned earlier, that BpALS (pALS anchor digested with BstXI) is better overall because it is longer and makes the optical tweezer operations a little easier and better.
pBR322 can be digested with EarI or SapI for use in our construct for DNA unzipping. I haven’t done any studies that show which method produces better results in terms of final yield, but I’m pretty convinced SapI will because you only have to perform one gel extraction. If you digest pBR with EarI you get two linear fragments. And according to separate analysis by Koch and myself the overhang that we need for the unzipping construct ligation is the longer piece (in the gel image linked above it is the brighter top band).
The benefit to following this method is you can perform a three piece ligation (essentially ligating the entire construct together in one shot) because the EpBR piece (pBR322 digested with EarI) can only ligate to the adapter, and the BpALS/BpRL piece can only ligate to the other end of the adapter (both adapters that I’ve been using work the same way).
And so today I’m doing the proper three-piece ligation reaction. The reaction setup and method can be seen in the table below: