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Linear Ligation

How can I ligate two or more linear fragments?

Open the Ligate [N] Fragments Dialog

To open the Ligate [N] Fragments dialog, click Actions → Linear Ligation → Ligate [N] Fragments... .

Specify the First Fragment File

Choose the file that contains the first fragment from the Source of Fragment 1: menu.

Specify the First Fragment

To specify the first fragment, 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 into the boxes at the upper right. Review the blunting, enzyme selection, fragment selection, and orientation.

Specify the Second Fragment File

To specify the source of the second fragment, click the Fragment 2 tab at the top of the dialog.

Choose the file that contains the second fragment from the Source of Fragment 2: menu.

Specify the Second Fragment

To specify the second fragment, click on the first enzyme site, then either Shift-click on the second enzyme site or drag to the second enzyme site.

Specify Any Additional Fragments

If necessary, repeat the steps described above to specify additional source files and restriction 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 flipping fragments, or blunting ends, in order to make the ends compatible for ligation.

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

Preview the Product

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

Name the Product

Type the name of the product, then click Ligate.

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 more precisely where the fragments were ligated.

To illustrate in History view where the ligations occurred, click the Ligate Fragments operation name.

Alternative Copy/Paste Approach

Alternatively, copy and paste can be used to populate the LIgate [N] Fragments dialog.

To select the first fragment to be ligated, click on the first enzyme site in Map or Sequence view, then either Shift-click on the second enzyme site or drag to the second enzyme site. Click Actions → Linear Ligation → Ligate [N] Fragments... . The Ligate [N] Fragments dialog will open to the populated Fragment 1 tab.

Switch to an open file containing the second fragment to be inserted. Select that restriction fragment, and then click Edit → Copy Restriction Fragment.

Switch back to the Ligate [N] fragments dialog. Click the Fragment 2 tab at the top of the dialog. Click Edit → Paste to populate this part of the dialog.

Repeat this process for any remaining Fragment tabs in the dialog.

When you are ready to ligate, type the name of the product, then click Ligate.

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