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.