User GuidesSnapGene User Guide Restriction Cloning and Linear LigationInsert Multiple Restriction Fragments into a Plasmid

Insert Multiple Restriction Fragments into a Plasmid

How can I insert multiple restriction fragments into a circular vector?

Golden Gate Assembly: SnapGene does not currently have a dedicated tool for simulating Golden Gate assembly. You can use Actions → Restriction Cloning → Insert Multiple Fragments to simulate Golden Gate cloning of vector + fragments with appropriate Type IIS enzyme sites (e.g. BsaI) at their termini.

Open the Insert Multiple Fragments Dialog

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

Specify the Vector File

In the Vector tab, click the Vector: dropdown menu and choose the vector file.

Alternatively, drag a vector file into the box.

Specify the Vector Insertion Site

Restriction fragments can be inserted at a restriction site in the vector or can replace an existing restriction fragment in the vector.

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

To select a restriction fragment in the vector, click on the first enzyme site, then either Shift-click on the second enzyme site or drag to the second enzyme site. Alternatively, type the enzyme names in the boxes at the upper right. Review the blunting, enzyme selection, fragment selection, and orientation.

Specify the Number of Insert Fragments

By default the Insert Multiple Fragments tool starts with two inserts.

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.

Specify the Insert Source Sequences

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

Manual Selection

  1. First, set the Fragment via the "Fragments" dropdown menu.
  2. Click the dropdown to choose the "Source of the fragment", or
  3. Click the link to browse and set the "Source" of the fragment.

Drag and Drop

Drag and drop one or more files into the Fragments panel.

If more than one sequence is added simultaneously then the "Order of Fragment" Dialog will automatically open. If required, use the "Reverse Fragment Order" button (with all or no sequences selected), or select sequences in the list and use the "Arrow" buttons to change the insert order.

Once the sequence order is set, click OK to continue.

Cut and Paste

First, set the Fragment via the "Fragments" dropdown menu, then copy a sequence from any compatible source file and paste to import it as the specified fragment source.

Specify the First Inserted Fragment

To specify the source of the first insert, 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, 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 the Second Inserted Fragment

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, 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 Inserted Fragments

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

Make the Ends Compatible

If cloning is not possible, the box at the lower right will be yellow and will explain what steps must be taken to proceed. Correct the problem by some combination of flipping vector or inserts, or blunting vector or insert ends to make the ends compatible for ligation.

In the illustrated example, the yellow box indicates that the ends are not compatible for ligation. This can be corrected by clicking the reverse directional arrows to flip the orientations of both inserts.

Preview the Product

To preview the product, click the Product tab. The box will turn green if you are ready to clone.

Name the Product

Enter an appropriate name for the product, then click Clone.

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 red and blue colors represent the multiple inserted restriction fragments.

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

To illustrate in History view where the insertions occurred, click the Insert Fragments operation name.