I am trying to put together the promoter, gene and terminator in pUC19 backbone. I followed the protocol of NEB in the Gibson mix. But I am not getting any colonies in my plate ( even blue colonies). I made adjustment already in antibiotic concentration, volume of Gibson mix in transformation and even molar ration of fragments and backbone yet still not working. Can you suggest any information or protocol for the Gibson experiment?
Since you don't have any background colonies,
I would suggest that transformation is not working.
Check your competent cells, if possible, switch to electroporation.
I would not worry about the [Amp] since bla resistance marker
can allow bacteria to grow on 30 times higher [Amp] than the
commonly used (100ug/ml).
Instead of the GIbson assembly, you may try to assemble your
insert from fragments by running 5-7 cycles of high fidelity
PCR - larger product formation should be visible on the gel.
Thanks Misha! What we are using here in the lab are home made chemically competent cells. And I assure you that they are really competent because we've been using it to other constructs for transformation. But if you suggest electroporation, then I will try to do it also. Do you suggest to use commercially available competent cells?
You should always have background colonies. I'm siding with Misha on this one.
Yes John, I know. I am checking my competent cells now if there is problem. I will also try the electroporation as what Misha suggested.
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