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Revision as of 14:17, 8 May 2015

Page is under construction.
iGEM HQ is currently working on updating this information for the iGEM 2015 competition.

Ligation and Restriction Digest Troubleshooting

Now that you know you transformation efficiency and it's above 1 x 108 CFU/µg DNA, we can work on other possible problems if you're still not getting great results from your cloning. This page is focused on common problems students have with ligations and restriction digests.

No colonies

If you know your cells are working well, there are a few common ligation problems that might be happening with your reaction.

  1. Uncut insert: It's possible that your insert was not cut well during your digest.
  2. Ligase didn't work:

Too many colonies

Sometimes with ligation reactions you can end up with a lawn of bacterial growth where its impossible to select a single colony. While you may think this means your reaction worked really well, it actually indicates a problem with your restriction digest.

  1. Uncut backbone: The most common cause of a lawn of bacteria after ligation is an uncut plasmid backbone. This makes your transformation in essence a plasmid transformation and you get far too many colonies on your plate.