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T-vector self-ligation?

  1. Dec 11, 2011 #1
    Hello!

    I conducted a transformation experiment and plated the bacteria on antibiotic agar with blue-white screening. The plasmid I used was a linearized T-vector. The insert with A overhangs would insert into the lacZ gene. If it is successfully inserted, white colonies should form. However, apparently it's possible to get blue colonies. I don't understand why. Can the T overhangs self-ligate somehow? Or would it be something in the preparatory steps for constructing the vector - say, the prep was not complete and some circularized plasmids remained or the t overhangs were not added successfully? I was also thinking that maybe the bacteria could remove overhangs?
    I included a circularized control, is it merely to cover the possibility of contamination? I personally don't think so, I had a 'blank' colony (no insert included but t-vector was), on which nothing grew. Although this doesn't actually tell me much.

    Anyone got any ideas.
    Many thanks.
     
  2. jcsd
  3. Dec 11, 2011 #2

    Ygggdrasil

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    How did you linearize your T-vector? For your blank control, did you perform the ligation reaction or did you just transform the linearized vector without a ligation step?
     
  4. Dec 11, 2011 #3
    Hi Ygg, thanks for the response. Thats the thing, I was simply given a linearized vector, no explanation, so I don't know the process for it. For the control, I subjeted iot to the ligation as if there was some insert, but I just didn;t include an insert in the mix. It went through all the same processes otherwise. he blue colonies have to be self-ligation, since its the only way for the bacteria to survive and the colonies to be blue, so it must be in the prep step that I din;t do. Can they even self-ligate once the overhangs are on?

    Thanks for the response.
     
  5. Dec 11, 2011 #4

    Ygggdrasil

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    Yes, the vector should not be able to self-ligate once it has the t-overhangs on it. The fact that you see no colonies in your blank is also a good indication that no self-ligation is going on. Blue/white screening is useful for distinguishing re-circularized vectors from plasmids containing inserts, and so the screening seems unnecessary because your vector is designed to prevent self-ligation.
     
  6. Dec 11, 2011 #5
    Thanks Ygg, it must be in the prep step then. I haven't been taught how that works so I wasn;t aware of the procedure. Thanks again!
     
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