Cloning Clipboard

Click Cloning can be used to duplicate or design cloning experiments in the lab. The Cloning Clipboard allows you to clone multiple fragments into a vector.

To perform a ligation of two fragments into a vector.

1. Open the sequence containing fragment A.
2. Select a restriction site on one side of fragment A, hold down SHIFT and click on a site flanking your fragment.The fragment will be highlighted. Note that for circular sequences you must select these sites in a 5' to 3' direction.
3. Click DIGESTThe fragment will appear in the Cloning Clipboard.
4. Open the sequence containing fragment B.
5. Again select restriction sites on both sides of fragment B.
6. Click DIGESTFragment B will now appear in the Cloning Clipboard
7. In the Cloning Clipboard click on the end of fragment A and hold the mouse button. Any compatible sites will be highlighted with a black dot within the end. Any incompatible sites will be highlighted with a grey dot (you can modify these in the next step).
8. Drag the end of fragment A to the compatible end of fragment B. The Ligation Dialog will appear.
9. Modify the ends or click LIGATE if the ends are compatible. A third fragment will appear in the Cloning Clipboard with both fragments
10. Select this fragment in the Cloning Clipboard
11. Open your cloning vector
    • If there is an unambiguous ligation possible (i.e. a single compatible site for each of your fragment's ends) then click LIGATE
    • If there are multiple choices of sites for one or more ends select one site, hold down shift and select the flanking site.. Click LIGATE
    • If one or more sites are not compatible you will be able to modify the ends in the dialog that will appear..
12. The Ligation dialog is shown. Either click LIGATE (or FLIP and LIGATE) or modify the ends and then click Ligate to clone your fragment into the vector.

Note that the Ligation Dialog is always shown following a ligation event. However, in the Cloning Clipboard if all sites are compatible then holding the option key whilst dragging two ends together (or clicking CIRCULARIZE) will bypass the Ligation dialog. If sites are not compatible the ligation dialog is always shown.

To quickly create a new sequence from a single fragment, just double click the fragment in the Cloning Clipboard.

You can copy a fragment from the Cloning Clipboard and paste into a graphical application, such as Illustrator. This makes it easy to create a graphical illustration of a cloning strategy. The fragments are copied at a fixed width size, irrespective of the actual fragment sequence length. For when you need to have greater control there is an alternative way of creating a sequence from a single fragment, resizing the window until it's the size you need, then copying that.

Related Topics.

How to digest and ligate fragments into a vector (video)

Performing Click Cloning

End modification

Gateway and TOPO cloning

Restriction enzyme

Resetting the origin of a circular sequence.

Automatic Restriction Enzyme Analysis