Currently, I'm working on cloning experiments using competent cells (E.coli--> DH5-alpha). I've performed ligation before and continued with transformation in competent cells. after incubation overnight, I got colonies, then I spread the colony in Amp+X-gal+IPT to perform blue-white screening. unfortunately, after 16 hours in 37 degrees condition, I checked, and I didn't found any colonies grown. i did twice and the results were the same. could anyone know what caused my colony not to grow at all? thank you for your answer.
From what you describe, the problem is one of two things:
1. The Amp in the plate you plated the transformation on was going bad/bad to begin with and the resulting colonies you saw were not actually Amp resistant so they did not grow on the Amp+X-gal+IPTG plates.
2. You picked a "satellite" colony from the initial transformation plate which was not Amp resistant to begin with so they did not grow on the Amp+X-gal+IPTG plates.
You should make fresh Amp plates and retransform your ligation and the resulting colonies should be Amp resistant. Be sure to keep the Amp plates away from light since Amp is broken down by light.
I am a little confused by the description of what you did. Why did you not directly plate the transformation of your ligation directly on Xgal + ampicillin plates?
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