Insert Multiple Restriction Fragments into a Plasmid

To open the Insert Multiple Fragments dialog, click Actions → Restriction Cloning → Insert Multiple Fragments....

In the Vector tab, you can specify the vector sequence via one of the following methods:

1. Click the Vector: dropdown menu and choose the vector file.
2. Use the "Click here" link to open the OS file browser.
3. Drag a vector file into the window.

To select a single unique restriction site in the vector, click the enzyme site name.

To select a restriction fragment for replacement, click on the first enzyme site, then either Shift-click on the second enzyme site or drag to the second enzyme site.  Alternatively, use the dropdown lists or type the enzyme names in the boxes at the upper right.

In this example the vector will be cut with XbaI and KpnI.

Review the blunting (fill in or remove overhangs), enzyme selection, fragment selection, and orientation.

Switch to the Fragments tab.

To set the number of inserts in the reaction, use the "+" or "–" buttons. The number of expected inserts are displayed in the Fragments tab header, 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.

There are a number of ways to specify the insert source sequences, you can use any combination of the following methods:

First, set the Fragment via the "Fragments" dropdown menu then:

1. Click the dropdown to choose the "Source of the fragment", or
2. Click the link to browse and set the "Source" of the fragment, or
3. Copy and Paste: Select an appropriate restriction fragment with (compatible ends) in a SnapGene file and paste into the window, or
4. Drag and drop one or more files into the Fragments panel.

If more than one sequence is added simultaneously by drag and drop then the "Order of Fragment" Dialog will open. If required, select sequences in the list and use the Move: controls to set the fragment order. Once the order is set, click OK to continue.

Select  "Fragment 1" via the "Fragments" dropdown menu.

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, user the dropdown lists, 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.

To specify the source of the second insert, select "Fragment 2" via the "Fragments" dropdown menu.

As for 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, user the dropdown lists, 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.

Repeat the steps described above to specify any additional source files and insert fragments.

If cloning cannot proceed then no product will be drawn in the Product tab, and the box at the lower right will be yellow with an explanation of what is required to proceed.

Correct the issue by some combination of flipping vector or inserts, or blunting vector or insert ends to make 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 clicking the arrows to flip the orientations of both insert fragments.

To preview the product, click the Product tab. The box will turn green if all ends are compatible.

Enter an appropriate name for the product, then click Clone to create a new Product file.

To display the product history colors in Map and Sequence views, toggle on the "Show colors" button in the side toolbar and use the dropdown to choose History Colors. Alternating red and blue colors represent each inserted restriction fragment.

In Sequence view, the history colors illustrate precisely where the restriction fragments were inserted.

In History view to highlight the inserts and where the insertions sit in the product, click the Insert Fragments operation name.