The insertion cloning function can be used to insert a linear fragment into a circular sequence so that a new circular product can be created. Insertion Cloning can be initiated from either the linear sequence or the circular sequence. When the insertion cloning is initiated from the linear sequence, only a circular sequence can be used as fragment 2. When the insertion cloning is initiated from a circular sequence, only a linear sequence can be used as fragment 2.

 

1. Select the sequence tab that will be used in the insertion cloning.
2. Select CREATE -> Insertion cloning

3. In the newly opened cloning window, the current sequence is set as Fragment 1. Select the restriction enzymes that will be used to digest the sequence. This can be done by selecting the enzyme from the enzyme list at the start and end sites, or by searching for the enzyme name in the enzyme list.

4. Click the Fragment 2 tab on the top of the window or Fragment 2 icon on the insertion window to move to fragment 2.

 

5. If there is no suitable sequence opened, the new sequence can be added by using the Import File function or Create DNA Sequence function. Please refer to the Load Sequence user guide for more detailed instructions.

 

6. If there is a suitable sequence opened, it will be set as Fragment 2 by default. If this is the sequence you want to use, go to step 8. If this is not the sequence you want to use, you can follow step 5 to add a new sequence.

7. If there are two or more suitable sequences opened, all suitable sequences will be listed in the fragment 2 dropdown. You can either select a sequence from the dropdown or follow step 5 to add a new sequence.

8. Select the restriction enzymes that will be used to digest the sequence for Fragment 2, as described in step 3.
   1. If the digestion ends are compatible, the enzyme will be shown in blue.
   2. If the digestion ends are not compatible, the enzyme will be shown in red and the “CLONE” button is greyed out.

9. When both ends are compatible, there is a preview of the ligation sites at the insertion window, and the product can be previewed in the product tab.

10. Press the “CLONE” button to make the ligation product. After the product name is entered in the pop-up window, press “CREATE” to create the product tab with the new product name.