Our desired insert is small (around 78 bp long). We have used 0.02 pmol (as per kit protocol)of desired insert for annealing with pET Xa/LIC Vector. Then we transform 1 µl annealing reaction directly to NovaBlue GigaSingles™ competent cells and kept the tube in ice for 5mints for incubation, then heated tubes for exactly 30 s in a 42°C water bath. After that we immediately placed tubes on ice for 2mintues and added 250 µl room temperature SOC medium to tube. Then incubated the tube at 37°C with shaking (100 rpm) for 60 min prior to plating on LB/SOC medium. But no colony was found on plate (50 µg/ml ampicillin containing medium) after 24hours of incubation at 37°C.

What may be the reason behind this? Is there any problem during recombination or transformation?

Any suggestions would be greatly appreciated. Thank you in advance.