We have been having issues with this certain plasmid for a while: we were trying to remove one gene (GFP) and replacing it with another. The digest of the original plasmid with BamHI and NotI looked perfect on an agarose gel, but we kept getting false positive clones with the original GFP insert, and nothing with the new insert. When we now sequenced the undigested plasmid, the sequence appeared as expected in the areas of the BamHI and NotI sites (see file attached). When we had the digested plasmid sequenced, however, there was an ambiguous nucleotide in each of the restriction sites (directly before the restriction cut site). Does this happen when sequencing restriction digested DNA?! Or would you think that there is a proportion of DNA in our plasmid prep that carries those mutations? Also, the sequencing run continued beyond this point, indicating that, in fact, the plasmid had not been digested – or at least not completely. I think that also makes it less likely that the mutations are introduced by the UV light to visualise the DNA in the agarose gel, for example.
Has anyone got ideas what's going on here or even better - a solution? At the moment we are re-transforming the DNA back into competent cells to plate them out and pick single colonies - just in case there is some low level of mutated plasmid that then overtakes everything in the cloning.
While I can't really tell you what is happening, I think your idea of retransforming your plasmids and making new DNA from isolated single colonies is appropriate. It could be that your current DNA prep just has a mixture of normal and mutant DNA populations.
It happens during the sequencing. Sequence your insert twice to check any kind of error repeat
While I can't really tell you what is happening, I think your idea of retransforming your plasmids and making new DNA from isolated single colonies is appropriate. It could be that your current DNA prep just has a mixture of normal and mutant DNA populations.
No, the UV light used to visualize the DNA cannot create mutations. UV light causes mutations only in living cells.
My guess is that you grew up another batch of cells to get more plasmid for your digest. Mutations naturally happen at a low level. Start by streaking out to single colonies and make a freezer-stock of a bacteria line that doesn't have this mutation.
Good luck!
Do you perform gel extraction after loading your digests on an agarose gel? This would solve your problem since any plasmid mutated at a restriction site would not be digested. You should not get any false positives at later stages either since theoretically you purify only the vector backbone.
I would try different cloning method which is the RF cloning(restriction free cloning) especially if you want to substitute specific gene by another one usually it works very efficient with me ,you can find all the details about the RF cloning here http://www.rf-cloning.org/. The server can design your primers very easy.
Thanks for your suggestions, everyone. We do a gel purification which made it all so puzzling, as the old insert clearly dropped out and the backbone band ran at the expected size. We'll see where the re-transformation and picking of clones gets us. I'll also check out the restriction free cloning, might be an alternative. Cheers
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