Cloning Tool

The in silico cloning tool allows you to computationally create restriction fragments and assemble them by ligation.

• It is accessed from the “Tools” menu in the Construct Editor

(link to screenshot 13)

Restriction fragments can be created by dragging

• Any vector, construct, element or group of elements from the Library
• Any construct, sequence element or group of elements from the Navigation Pane
• Any construct, sequence element or group of elements from the Workspace Pane

into the Input Pane (label in screenshot 13)

DNA is digested in silico by choosing one or more restriction sites from the Sites Pane (label in screenshot 13)

• Restriction sites can be selected from all possible sites by
  • typing in the beginning of the restriction enzyme name in the box at the top of the “search all” tab
  • clicking on the sites of interest
• Restriction sites can be selected from one of your lists of managed lists of sites by
  • selecting the “my sites” tab
  • selecting the list name from the pull down menu
  • clicking on the sites of interest
• Once the sites of interest have been selected, click on the “choose” button at the bottom of the Sites Pane (label in screenshot 13)

• Once one or more restriction sites have been chosen, the sites will be shown
  • marked as lines on the graphical map in the Input Pane (label in screenshot 13)
  • as site names and positions in the Digestion Pane (label in screenshot 13)
    • when the mouse hovers over a site on the map in the Input Pane, it is highlighted in the Digestion Pane, and vice versa.
• A fragment can be cut by selecting the restriction sites that flank it, either by
  • clicking on the site in the graphical map, or
  • clicking on the site in the Digestion Pane
• “Cutting” the DNA will cause a segment of DNA to drop out of the fragment donor
  • Any “cut” segments of DNA may be dragged from the Input Pane to the Ligation Pane (label in screenshot 13) for assembly into the final construct.

(link to screenshot 13)

• “Cut” fragments of DNA that have been dragged from the Input Pane to the Ligation Pane (label in screenshot 13) are displayed
  • with their 5' and 3' overhangs shown
  • elements within the fragments can be seen by hovering the mouse over each element: the element will be highlighted in the Navigation Pane
• Each fragment becomes a single group, named after its construct and location of origin.
  • The fragment also retains all of its internal structure of groups and elements

• Fragments can be rearranged by dragging them up or down in the Ligation Pane
  • Prior to the ligation step, the right overhang of each fragment must match the left overhang of the following fragment.

• Once the fragment overhangs all match each other, press the “Clone” button at the bottom of the Ligation Pane
  • The resultant construct can be dragged into the Design Toolbox or Vector Library in the Library, or as a new construct in the Navigation Pane of the Construct Editor