What is the difference between T4 DNA Ligase and E.coli DNA Ligase?
The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.
Which T4 DNA Ligase protocol do you recommend when ligating an insert containing one cohesive (sticky) end and one blunt end?
For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends. The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours).