How do you troubleshoot a ligation?

Troubleshooting Tips for Ligation Reactions

1. Add controls, including vector alone, insert alone and uncut vector.
2. Vary the molar ratio from 1:1 to 1:10 vector:insert (1:20 for short adaptors).
3. Insert or plasmid should have a 5´ phosphate.
4. Use fresh buffer as the ATP or DTT may degrade over time.

What are the steps of a ligation reaction?

The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing.

How much ligation product do I need for transformation?

Transformation. Add between 1-5 µl of ligation mixture to competent cells for transformation.

What is the purpose of ligation and transformation?

The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.

Why is my ligation not working?

Ligations only fail for one of three reasons. First, your DNA ends are not compatible, Second, you have a chemical inhibitor or damaged DNA (e.g. excess UV treatment) that blocks successful ligation. Third, your vector has high background (incomplete digestion), and you’ve already ruled this option out.

How do you know if ligation worked?

Try including a positive control of ligation (vector digested with one enzyme, gel purified, and re-ligated). If that gives you colonies (and the same reaction mix without ligase does not), your DNA ligase is working.

How can you prevent self ligation?

THE MOST BASIC STEP FOR PREVENTION OF SELF LIGATION IS CUTTING THE INSERT AND VECTOR WITH 2 DIFFERENT RESTRICTION ENZYMES, GENERATING FRAGMENTS WITH 2 DIFFERENT RESTRICTION SITES. Removing 5′-phosphate groups from the vectors using phosphatases (e.g. alkaline phosphatase), prevents self- ligation.

How much ligase to use for bacterial transformation?

For transformation with ligated DNA, ligase and other reaction components carried over can reduce transformation efficiency. For heat shock, do not use more than 5 µL of ligation mixture for 50 µL of competent cells.

How can you tell if a ligation reaction is successful?

Any colonies formed from transformation of this control will be the result of undigested or re-ligated vector, and subtracting the number of colonies formed from this control from the number of colonies formed from the ligation will give an idea of whether the ligation reaction has been successful.

How much ligation mixture do you need for electroporation?

For heat shock, do not use more than 5 µL of ligation mixture for 50 µL of competent cells. For electroporation, the DNA should be purified from the ligation reaction prior to transformation.

What is the problem with DNA ligation in E coli?

If transformation of E.coli with the undigested parent vector leads to no colonies on the plate, the problem is either in the transformation procedure, the competent cells, or antibiotic selection. Thus, the next step would be to try new cells, or review your transformation procedures.