Click Cloning: End modification

MacVector will allow you to manipulate the ends of restriction fragments before joining them together. An interface lets you cut back or fill in each end of the source or target molecule before ligation (e.g. Klenow blunting). This will allow you to fill in 5' overhangs, and digest 3' and 5' overhangs on either the vector or the insert. MacVector will show you when it is possible to ligate the insert into your vector as compatible ends will be highlighted in red or green (these colors are user configurable. You can also flip the insert to change the orientation of the insert when it is ligated into the vector.

To modify the ends of a fragment and/or vector

The Ligation dialog is displayed following a ligation showing the ends of both vector and insert. Any compatible ends will be highlighted in either red or green and Ligate will be disabled.

Choose one end of the fragment and either fill in or cut back the fragment and or vector end until both are ends are compatible. The dialog indicates when they are compatible by highlighting the fragment ends and corresponding vector ends in red or green.
Repeat this for the other end of the fragment if required. Once all four ends are compatible Ligate will be enabled.
Click LIGATE to ligate your insert into the vector

Note that even if the ends are not compatible you can force the ligation by clicking LIGATE ANYWAY

Click Cloning: End modification

To modify the ends of a fragment and/or vector

Related Topics.