Ligation - Perserverance strikes back

After one month of ligation experiments, two out of three constructs were successfully subcloned into our lab's vector. For the last construct, I am using Vladimir's clone to save time.

This period had been frustrating and trying where after repeating so many experiments, the inserts were not ligated to the vector. I had tried different T4 DNA ligases from Roche, Promega and New England Biolabs. The ligation reaction mixtures had been incubated at room temperature, 16 degree celsius and 4 degree celsius. Incubation time tried were as short as 5 minutes to overnight.

Nearing the end of one month, I was feeling down and self-doubt set in. Many thanks to my friend, TuanLeng who encouraged me and discussed with me the possible mistakes in ligation. I perservered and for the last ligation experiment, I finally determined the vector and insert concentrations and calculated the volumes to react for giving a molar ratio of 1 (vector) : 3 (insert). Background and negative controls were set up. The background control was the vector without any insert while the negative control had insert only without the vector. Colonies from the background control helped to gauge the ligation efficiency. No growth should be observed on the negative control plate.

Lo and behold, when I saw the gel, I was happy that there were bands corrresponding to the sizes of my inserts. Their identities were confirmed from DNA sequencing.

These are the possible mistakes for failure in ligation experiment.
1. Ligase is usually dissolved in glycerol. Presence of >10% glycerol in the reaction mixture may inhibit ligase. In 10 ul of reaction mixture, 0.9ul was added instead of 1ul and the pipette tip was drained off excess ligase that might be adhering outside the tip.

2. Make sure the ligation buffer is new or used one is always kept on ice and stored at -20 degree celsius immediately after use since ligase requires ATP for ligating.

3. Vector and insert must be rid of ethanol since ethanol inhibits ligase too.

4. Calculate the molar ratio according to product information sheet. My mistake was that I followed others' recipes blindly. Alternatively, look at the formula below by Cranenburgh (2004) for calculating the volumetric ratios required in a ligation reaction. The T volume (represents the combined volume of vector and insert solutions e.g. T is 8ul in a 10ul reaction with 1 ul each of ligase and 10x buffer) can be scaled up accordingly.











Reference
Cranenburg R.M. (2004) An equation for calculating the volumetric ratios required in a ligation reaction. Appl. Microbiol. Biotechnol. 65, 200-202.