User:Anthony Salvagno/Notebook/Research/2009/09/30/Brian's Ligation Results

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User:Brian P. Josey/Notebook/2009/09/30 contains the results of the gel from the ligation. Also I have some notes on there about what we are seeing.

Commentary

If you look at the image, there are definitely 3 distinct bands in there. Under proper ligation conditions this would mean a successful ligation. In this case it does not. Quick Note: The 2 lanes not the ladder are both the same product split into two lanes. The upper band is 2.5 kb and that is definitely the plasmid piece. The lower band is about 1.1kb and that is definitely the anchor. Why is there another band? Take note of the fact that the really dim band seems to be just above 1.5kb. Check out this image:


I cut the slice (upper band) from this gel and I think I may have gotten some of the lower band (1.8kb) in my slice. Although now I am skeptical because that band looks a lot like 1.5kb not like 1.8kb. Maybe there could also be some kind of weird concatamers with the adapter duplex, but I find that unlikely.

In any event the ligation failed and I think there are 2 possible problems:

  1. no BstXI digestion of anchor
  2. no T+B annealing

Maybe the T4 Ligase is choking the chicken as well, but who knows. Koch, any thoughts?

Steve Koch 02:49, 1 October 2009 (EDT): After just reading your other comments, I came to same conclusion that it's just leftover from the gel extraction. I do have thoughts, and we should talk about them tomorrow. Basically just systematically approaching this problem. I know you can get it soon. One way of looking at my thoughts is I want to do more planning and less reacting to results. A trap that I commonly fall into.
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