The linear ligation function can be used to link two linear fragments together to create a new linear product. The linear ligation function can be initiated from linear sequences only. This function is not available for the circular sequences.

 

1. Select the sequence tab that will be used as the 5’-end of the product.
2. Select CREATE -> Linear ligation

3. In the newly opened ligation 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. The starting enzyme is optional.

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

 

5. If the sequence used in step 1 is the only linear sequence opened, no sequence will be set as fragment 2 by default. The sequence of fragment 1 is listed in the fragment 2 dropdown. A new sequence can be added by using the Import File function or Create DNA Sequence function. Please refer to the Add Sequence user guide for more detailed instruction.

 

6. If there are two linear sequences (including the fragment 1 sequence) opened, the second linear sequence 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 more than 2 linear sequences opened, no sequence will be set as fragment 2 by default. All linear 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 of Fragment 2, as described in step 3.
   1. The end digestion of fragment 2 is optional
   2. If the start digestion of fragment 2 is not compatible with the end digestion of fragment 1, the enzyme will be shown in red and the “LIGATE” button is grey out.

1. 3. If the start digestion of the fragment 2 is compatible with the end digestion of the fragment 1, the enzymes will be shown in blue

 

9. There is a preview of the ligation sites in the insertion window and the product can be previewed in the product tab.

10. Press the “LIGATE” 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.