Insert a Fragment into a Plasmid

How can I insert a restriction fragment into a circular vector?

A restriction fragment can be inserted at a restriction site in the vector, or it can replace a restriction fragment in the vector.

Specify the Insertion Site

Open the vector file.

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

To select an existing 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.

In the above example, a compatible insert will replace an existing BamHI-EagI fragment in the pSEC7-EGFP vector.

Open the Insert Fragment Dialog

Click Actions → Restriction Cloning → Insert Fragment... .

Preview the Vector

  1. Review the enzyme selection and vector blunting (if required).
  2. Confirm the correct fragment is selected for replacement by the incoming insert.
  3. If required, click an arrow to set the "Orientation of the vector" fragment with respect to the insert .

SnapGene version 6.0 and later allow the orientation of the vector to be reversed during cloning operations.

Specify the Fragment Source Sequence

Switch to the Fragment tab.

There are a number of ways to specify the fragment source sequence, you can use any one of the following methods:

  1. Choose the file that contains the insert via the Source of Insert: dropdown.
  2. Drag and drop a sequence file into the Fragment panel.
  3. Click the "Click here" link to browse and set the "Source of Fragment".
  4. Copy a sequence from any compatible source file and paste to import it as the fragment source.

Specify the Insert Fragment

To specify the fragment to be inserted:

  1. Click on the first enzyme site, then either Shift-click on the second enzyme site or drag to the second enzyme site.
  2. Alternatively, use the drop-down menus or type the enzyme names in the boxes at the upper right.
  3. Review and if required, reverse the orientation of the insert fragment.

In the above example the sites BamHI and NotI are selected. The insert-based NotI and vector-based EagI have compatible cohesive ends (5'-GGCC overhang).

Preview and Create the Product

Switch to the Product tab. The overview at the bottom summarizes the reaction.

The warning box at the bottom right, if green, indicates you are ready to clone.

Enter an appropriate name for the product sequence.

Click Clone to create a new product sequence file.

Click File → Save to save the new product sequence to an appropriate location on your computer.

If cloning is not possible, the warning box will be colored yellow and will explain what steps must be taken to proceed.

View the Product History Colors

In the product file, click the "Show colors" button in the side toolbar and select "History Colors". The red color represents the inserted restriction fragment.

In Sequence view, the history colors illustrate precisely where the restriction fragment was inserted.

In History view, to highlight the insertion fragment location, click the Insert Fragment operation name.

Alternative Copy/Paste Approach

Copy and paste can be used to populate the Insert Fragment dialog.

To clone by copy & paste:

  1. Open the file for the insert, select the restriction fragment, and click Edit → Copy Restriction Fragment.
  2. Open the vector file, select the insertion site or the restriction fragment to be replaced, and click Edit → Paste. The "Insert Fragment" dialog will automatically open to the populated Product tab.
  3. Type the name of the product, then click Clone.