How to design a digest to screen minipreps after a ligation.

By Chris | Published: June 12, 2024

MacVector’s Agarose Gel tool can be used to quickly design a restriction digest to screen minipreps following a ligation. 

(View full size on website…)

Replicate your ligation in MacVector.

  • Select the two sites, for subcloning your targeted gene, and click DIGEST.
  • Drag the digested fragment from the Cloning Clipboard to your vector
  • click LIGATE.

Create your agarose gel with the correct insert and a vector only lane.

  • Go to FILE > NEW > AGAROSE GEL
  • Open your sequence and switch to the MAP tab.
  • Drag a restriction site (or sites) that digest within the fragment and also in the vector to the Agarose Gel window.
  • Open up your original cloning vector.
  • Drag the same site(s) to the Agarose Gel window.

Undo the ligation, and repeat with the wrong orientation.

  • Switch back to the ligated sequence, and use UNDO to remove the ligated fragment.
  • Switch back to the Cloning Clipboard.
  • Drag the same digested fragment from the Cloning Clipboard to your vector.
  • Hold down [OPTION] and click LIGATE.
  • Switch to the MAP tab.
  • Drag the same site (or sites) that digest the fragment and the vector to the Agarose Gel window.

Now you will end up with an Agarose Gel with three lanes: A lane with empty vector, a lane with the insert in the correct orientation, and a lane with the insert in the wrong orientation. Now it’s easy to screen your minipreps, as you know the gel bands of a correct miniprep before you’ve even loaded it on the gel!

This entry was posted in Tips and tagged , , . Bookmark the permalink. Both comments and trackbacks are currently closed.