Directional Topo cloning, no colony
I am having a problem with pENTR-d-Topo cloning. I have spent one month doing topo cloning but can not get a single clolony. Here is how I do:
I did my PCR using phusion HF polymerase and extacted the blunt end PCR products, then check it with digestion, everything works well, then I follow the d-topo cloning protocol of Invitrogen, including control, vector insert ratio gradient at 1:1, 1:3, however, after the transformation, I can not even get a single colony, I tried so many times, all the plates are just blank, does anyone have any idea what is going on?
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