Self-ligation without DNA ligase possible !! How??
I have a clone wherein the insert of 1.5 kb (NdeI-NdeI) was in reverse orientation.
Hence, I cut out the insert by doing NdeI digestion, heat inactivated the enzyme and did a transformation into XL-1blue cells. Once I started doing transformation, I realized that I had not set up a ligation reaction for re-annealing of the digested fragments. Nevertheless, I continued with what was evidently a lost cause. However, I got a couple of colonies on the plate and upon screening found 2 with the insert in the correct orientation. Lucky. However, what I do not comprehend is how did the digested fragments ligate when I never added any DNA ligase? I thought ATP and ligase are absolutely required for the phosphodiester bond formation and hence religation. Could anyone please shed some light on it?
E.coli cells you transformed your cells into contain DNA ligase, otherwise your cells would be unable to repait DNA brakes and would be dead. Depending on the genotype of the strain used, the cells also lack DNA-degrading enzymes, which futher facilitated the reaction
Thanks a lot.