priciple of cloning

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    • #8307
      piefke
      Participant

      Hi
      I´m trying to create a clone and have some ligation problems. And while I am thinking about the reasons for these problems and looking on my gels- I have one question.
      When I run a gel with my ligation-mix there is a lot of smear. And it seems to be logical because my cut insert can ligate with another cut insert and the cut
      vector can ligate with another cut vector…. and so on…. so there are many chains of consecutive dna-fragments of different sizes at the end of a ligation. Is this right?
      But when that is the truth: how can a ligation and a cloning work?? I only want to ligate one insert with one vector. I know it must work, because a lot of people clone successfully- but I do not understand how it works…. 😕
      Perhaps it is a stupid question…. but I would be happy about an answer…..

    • #76487
      mith
      Participant

      Actually there are some size limitations, so while the insertion of target dna into the vector is favorable, vector to vector is much more unlikely. You should also lookup what the following steps are in cloning. *hint, you have to screen using antibiotics most likely.

    • #76489
      SororSaudade
      Participant

      Maybe I didn’t get your question… but why don’t purify the fragments you want before the ligation?
      Like mith said… you can also select the plasmid you want after ligation and transformation, but that’s very time consuming (though you sometimes have to do it).

    • #76490
      blcr11
      Participant

      You may or may not be able to see a band when you run a ligation mix out on a gel. You are more likely to see the smear you describe. The “trick” to ligation is to use the correct ratio of insert to vector DNA and to use a volume which is neither too small or too large. For instance, Novagen’s sticky-end ligation protocol using T4 DNA ligase calls for using 0.03 pmol of digested vector and 0.2 pmol of digested insert DNA (or roughly a 6.7-fold molar excess of insert over vector) in a 10 microliter final volume for 2-16 hours at room temperature. You want the vector-insert end-recognition to be a facile reaction, so you want to use high concentrations of ends. But you also want recircularization to be likely too, and that is favored by dilute solutions. If you use too high a concentration of ends, you will reduce the yield of recombinant plasmid because most of the mix will form large concatemers. But if you use a really dilute condition to favore reclosure, then the main reaction is recircularized vector without insert if the vector ends are compatible, or nothing at all if the vector ends are not compatible. Typically, ligation volumes are in the range of 10-20 microliter. In a few cases (LIC cloning reactions, for example, but these are probably special cases; there is no ligase involved and the vector ends are not compatible) the volumes can be as small as 5 microliters. You then use 0.5-1 microliter of the ligation mix to transform competent bacteria. Concatemerized inserts-only can’t grow. Antibiotic selection should allow the growth of anything with functional vector in it—which hopefully will be recombinant plasmid with only one copy of the target DNA sequence inserted, though you may sometimes get multiple copies of insert, or recircularized vector-alone, so you need to screen your clones for the presence of the correct insert.

    • #76550
      Duke
      Participant
      quote piefke:

      Hi
      I´m trying to create a clone and have some ligation problems. And while I am thinking about the reasons for these problems and looking on my gels- I have one question.
      When I run a gel with my ligation-mix there is a lot of smear. And it seems to be logical because my cut insert can ligate with another cut insert and the cut
      vector can ligate with another cut vector…. and so on…. so there are many chains of consecutive dna-fragments of different sizes at the end of a ligation. Is this right?
      But when that is the truth: how can a ligation and a cloning work?? I only want to ligate one insert with one vector. I know it must work, because a lot of people clone successfully- but I do not understand how it works…. 😕
      Perhaps it is a stupid question…. but I would be happy about an answer…..

      Well, I hope I understood you correctly. You weren´t too specific on what you did when observing the problem:
      You cut your vector with 2 enzymes and also your insert: By this only one insert can be ligated into the vector as the 5´ends of the insert don´t match the 3´ends of the inserts and thereby can not be elongated to mutimers. Also no religation of empty vector is possible. But a prerequisite of every cloning step is always the purification (e.g. on an agarose gel) of the cut pieces.
      If you cut only with one enzyme you theoretically could get multimers but it´s much more likely that the empty vector just religates and you get only empty vector (this reaction is easier for the ligase to catalyse than elongation with 2 or more fragments as the ends of the vector are in closer and more stable proximity as flowting pieces in the ligation solution). Therfore you need to dephosphorylate the vector first. This prevents religation and vector multimers.
      It is always possible to get still multiple insertions of insert later but the chances are very low as it is difficult for the ligase to "carpure" an insert from the solution ad ligate it with the vector. once one end is ligated it is always way more easy and likely that the other end of that insert will be ligated with the same vector just because of proximity and time beeing close to each other.
      For example the chances of a successful triple ligation (2 inserts cut with 3 enyzmes to be liagted into one vetor) are much worse than with a normal ligation with one insert ligated in one vector cut with 2 enzymes. But it is possible.

    • #76557
      piefke
      Participant

      hi
      Thank you for your explanations. Now I see that a cloning procedure is much more complicated than I thougt before… 😉

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