If desired, a TOPO cloning vector may be constructed manually and added to the Cloning Vector Catalog prior to beginning the cloning workflow. To make the vector recognizable for directional TOPO cloning, a specific overhang must be created at the 3’ end of the forward strand.

  1. Use File > Open to open the file or SeqBuilder Project containing the vector sequence.
  1. To the 3’ (right) end of the vector sequence, add the bases cacc.
  1. Choose Cloning > Change Overhangs.

    In this dialog, you can use the Trim Blunt or Fill In buttons to simulate modification of overhangs for incompatible ends, or manually manipulate the overhangs by dragging the arrows located above and below the two strands. The Revert button lets you return to the default overhangs.

    1. On the left, make sure the pair is blunt, i.e. does not contain any overhangs. On the right, drag the top arrow to the left to “mask” the bases you just added (cacc). This results in a gtgg overhang on the bottom right.



    2. Click Change.
  1. Choose File > Save.
  1. Add the new vector to the Cloning Vector Catalog so it can be used with future projects.

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