Question

A researcher had a lot of colonies on the positive control plate after transformation but not a single colony on the ligation reaction plates.  What are some ways the researcher can alter the ligation conditions to improve the chance of success?

Answer 1

Positive control plate means here is that bacteria is transformed with only circular plasmid (no clone gene) only.

Ligation reaction plate meaning here bateria is transformed with cloned plasmid (clone gene) in which no colony formed it means no ligation occur between plasmid and gene of interest.

Although above result suggest that plasmid has gone for 100% effieciently digested with restriction enzyme so to get ligation done, we need to change ligation parameter like 23C incubation for overnight or 4C incubation at overnight or 37C for 1 hrs incubation.

So we need to try several hit and trial conditions to obtain ligation.

Hope it's clear..thanks