Inserting DNA Fragment into Plasmid

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    • #14158
      NiceOnTheIce
      Participant

      Scenario: You digest DNA into fragments and cut plasmids at a specific site, then mix them together and add ligase to hopefully insert a DNA fragment into a plasmid and close it up. How do you ensure that the DNA is inserted in the proper orientation (not backwards?) Assume the DNA has sticky ends and not blunt ends.

      This is just a summary of a question I have to answer, but knowing that the DNA fragment has the same number of bases sticking out of its 5′ end on both strands (the complimentary strands are shifted, which creates the 5′ sticky ends) how DO you make sure it inserts properly? Is this a trick question?? The way I see it, the DNA fragment could be inserted either way because the strands are complimentary, which means the bases on their sticky ends are exactly the same, and would H-bond with the plasmid bases no matter how it was inserted.

    • #102373
      JackBean
      Participant

      well, you wrote only summary of your question, so I don’t know, whether there were some more informations, but you can cut it with two different REs, which produce different overhangs and thus there’s higher chance to be it inserted correctly.
      Or you can use one RE producing 5′-overhang and other producing 3′-overhang 😉

    • #102379
      NiceOnTheIce
      Participant

      The full question is this:
      "You are a summer research student in a molecular biology research lab. You are asked to insert the "stresslessontests" gene into a plasmid. It is important that the gene is inserted in the proper orientation so that a functional protein can eventually be made after the recombinant plasmid is transformed into E.coli. Using detailed diagrams accompanied by a written narrative, illustrate and describe the steps you would follow to create this recombinant plasmid. Be sure to include all of the enzymes needed to do this (be specific), as well as any relevant data sequence requirements. Show the intermediates of the process (i.e. digest fragments with sticky ends) as well as all of the products resulting from this process."

      I summarized it because I only need help with part of the question: What do you use to ensure that the gene is inserted into the plasmid in the proper orientation?

    • #102382
      JackBean
      Participant

      well, then as I said, use two different REs with different overhang sequences 😉

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