User:Anthony Salvagno/Notebook/Research/2009/09/08/Ligation Planning for capped DNA

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Done so far

So far I have ligated 1ug of pBS DNA with unknown quantity of cap DNA. There is about 600ng of DNA in 2 tubes and I think I should combine them in one tube and concentrate it. I can run a column, but I really don't know how since it is in elution buffer already. I guess I should just mix it with cleaning agents (from Qiagen kit) and then elute again into 30uL EB.

Steps to take

  1. Ligate capped DNA to unzipping construct.
    • Can do this in 2 different ways:
      1. Ligate anchor to T+B fragment and then ligate construct to capped DNA
      2. 3-piece ligation of anchor, T+B, and capped DNA. This way I have to use a little of T+B so as to not quench both anchor and capDNA so that the 3 pieces can form one strand.
  2. Finish Tweezers
    1. Adjust the steering optics for optimal tweezing
    2. Put in QPD


When combining the two tubes somehow I lost the DNA, so I have to do a new digestion of pBS with Sap and another reannealing. I also think that I will digest the NotpBS with SapI so that I can ligate that onto the SapI overhang of the unzipping construct. In either case I need to do some ligations of anchor to T+B, maybe to do that and a 3-piece ligation.

Upon further review, I cannot digest the NotpBS with SapI. This will create two pieces of dsDNA a small fragment with the MCS in it and a large fragment with the rest of the plasmid. The small piece has the overhang that gets ligated to the construct. While we can do the ligation, I feel it is not worth unzipping the little DNA fragment. Sap cap will have to do.