Troubleshooting/Ligation
iGEM HQ is currently working on updating this information for the iGEM 2015 competition.
Ligation and Restriction Digest Troubleshooting
Now that you know your 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 researchers have with ligations and restriction digests.
Negative and Positive Controls
It's easy to forget or skip controls when you're doing restriction digests and ligations. I strongly urge you to always run controls for these reactions as they will give you a lot of information and allow you to more easily troubleshoot your cloning problems. By running a few extra reactions, you can potentially save yourself many hours and days worth of troubleshooting later. Due diligence when it comes to cloning is well worth the effort, I promise you!
When comparing your backbone + insert ligations, you ideally want to see many more colonies than from the backbone control ligation (cut backbone with ligase below).
| Control | Ligase | Results |
|---|---|---|
| Cut backbone | no (-) | Colonies will give you an idea of the background due to uncut vector due to inefficient restriction digest of the backbone |
| Cut backbone | yes (+) | Colonies will give you an idea of the background due to the re-circularization of cut backbone |
| Cut insert | yes (+) | Colonies indicate contamination of intact/uncut plasmid in your ligation and/or transformation reagents |
Generally, a 3:1 insert:backbone ratio will work well for two-part BioBricks assembly. You can
New England Biolabs has a useful online Ligation Calculator that can help you determine how much of your insert DNA you need to add in various gram values. In order to use this tool, you need to know the length of your Insert DNA, the length of your Vector DNA (or backbone), and the amount of your Vector DNA you have for your reaction (labeled Vector DNA mass). This is most often entered as nanograms or micrograms. You can set which scale you want to work in (example: nanograms or micrograms) and it will give you a series of different ratio outputs you can try to optimize your ligation if the 3:1 ratio fails for you.
Few or no colonies
If you know your cells are working well, there are a few common ligation and digest problems that might be happening with your reaction.
- Uncut insert: It's possible that your insert was not cut well during your digest.
- 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.
- 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.
- To check for this, run a DNA agarose gel with your cut plasmid alongside a lane of uncut plasmid. Your uncut plasmid should appear to run smaller due to the supercoiled nature of uncut plasmid. Your cut plasmid should run higher since it's no longer supercoiled after being cut by the restriction enzyme(s).
Also, for most BioBrick assemblies, when you cut open the backbone you are removing a piece of DNA from the cloning site. This will create two (or more) bands to appear on your gel.