My insert DNA (size 1.5kb) was gel purified sample with a good concentration (100ng/ul , 260/280-1.94 and 260/230- 1.90). I used pGEM-T easy vector (size-3kb) for ligation and followed manufacturer's protocol. I have tried overnight incubation (4 degree) and 2 hours RT incubation. After the transformation experiment I found only 3 blue colonies in the plate (Amp+/LB media and spreaded x-gal (100mg/ul)). I don't know where I made a mistake.
Anyone suggest me how to overcome this issue?
P.S. I used DH5a competent cells for transformation. I have done ligation and transformation with different insert previously and i got a good result, so this time I hope there is a problem with ligation.
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