How can I ligate two or more linear fragments?
Open the Linear Ligation Dialog
To open the Linear Ligation dialog, click Actions → Linear Ligation.
Specify the Number of Fragments for Ligation
By default the Linear Ligation tool starts with two fragments.
To set the number of fragments for ligation, click the "+" or "–" buttons. The number of expected fragments are displayed in the Fragments tab, and listed in the "Fragments" dropdown menu.
For larger numbers of fragments, click the drop down and choose "Number of Fragments", in the dialog, enter the number of fragments then click OK.
Specify the Fragment Source Sequences
There are a number of ways to specify the fragments, you can use any combination of the following methods:
First, set the Fragment via the "Fragments" dropdown menu.
- Click the dropdown to choose the "Source" of the fragment", or
- Click the links to Browse and set the "Source" of the fragments, or
- Copy and Paste: Select an appropriate restriction fragment with (compatible ends) in a SnapGene file and copy then paste into the window, or
- Drag and drop one or more source files into the Fragments panel.
If more than one sequence is added simultaneously then the "Order of Fragment" Dialog will automatically open. If required, select sequences in the list and use the "Move: controls to change the fragment order. Once the sequence order is set, click OK to continue.
Specify the First Fragment
To specify the source of the first fragment, select "Fragment 1" via the "Fragments" dropdown menu.
To specify the fragment, click on the first enzyme site and then either, Shift-click on the second enzyme site, or drag to the second enzyme site.
Alternatively, use the dropdowns, or type the enzyme names into the boxes on the right.
Review insert blunting (fill in or remove overhangs), enzyme selection, fragment selection, and orientation.
Blunting (or polishing) simulates enzymatic backfilling or removal of single-stranded overhangs - see Blunting.
Specify the Second Fragment
To specify the source of the second insert, select "Fragment 2" via the "Fragment" dropdown menu.
As with the first fragment, to specify the insert click on the first enzyme site and then either, Shift-click on the second enzyme site, or drag to the second enzyme site. Alternatively, type the enzyme names into the boxes at the upper right.
Review insert blunting (fill in or remove overhangs), enzyme selection, fragment selection, and orientation.
Specify Any Additional Fragments
If necessary, repeat the steps described above to specify additional source files and fragments.
Make the Ends Compatible
If ligation is not possible, the box at the lower right will remain yellow and will explain what steps must be taken to proceed. Correct the problem by some combination of reordering fragments, flipping fragments, or blunting ends, to the ends compatible for ligation.
In this example, the yellow box indicates that the ends are not compatible for ligation. This can be corrected by reordering the fragments so the compatible BamHI overhangs are adjacent.
Preview and Create the Product
To preview the product, click the Product tab. The box will turn green when you are ready to ligate.
Type the name of the product, then click Ligate. to create a new "Product" file.
View the Product History Colors
To turn on the product history colors in Map and Sequence views, click the "Show colors" button in the side toolbar. Alternating black, red and blue colors represent multiple ligated fragments.
In Sequence view, the history colors illustrate precisely where the fragments were ligated.
To illustrate in History view where the ligations occurred using the History colors, click the "Ligate Fragments" operation name.
