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I`ve been trying to clone a fragment (about 1.6 kb) using pGEM system, unfortunately it was not succesful. Then, I moved to pJET, and guess what? Again, very low transformation efficiency. Now I am moving to pENTR. However, I am think about why I am struggling to clone this fragment. I have tried to increase insert:vector proportion; plate a higher # of e. coli cells; left my ligation reaction overnight, but nothing is very helpful. Does anyone have any guess or suggestion about it? Does anyone think that the fragment is too long? Thanks :)

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The fragment size itself shouldn't be a problem in principle.

Cloning has many steps, so anything you didn't specifically test could be causing problems. If different vectors are also not the problem, you should probably look at your experimental steps:

  • One problem can often be residual EtOH from cleanup steps, which will make your ligation and transformation very inefficient.
  • You should also test your competent cells with another plasmid to be sure, that they are still viable
  • Have you checked that all fragments run at the correct size on a gel? You might have star-digestion or bound enzymes that cause problems. Additionally you could also check the ligation product on a gel (though you won't be able to use that for transformation), to see if the reaction works correctly
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  • $\begingroup$ Hi Nicolai, thank you so much for your suggestions! I'll test it! Best regards, Matheus! $\endgroup$ – Matheus Nogueira Jun 13 '17 at 17:23

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