I made a new plasmid by gateway cloning, and got the positive colonies by restriction enzyme digestion successfully. but then I transformed into Agrobacteria EHA105, when confirming transform back into E coil, the plasmid changed by restriction enzyme digestion, then I transform the positive plasmid confirmed before into regular E coli (EPI300), pick the colonies, the plasmid changed too, then I reculture the E Coli stock I made at the first time, pick the colonies, the plasmid changed too. But the first positive plasmid still right after digestion. Does anybody know what happened?
Is it possible that the positive colony you selected at the beginning was in fact mixed carrying two different plasmids and your subsequent steps had picked the wrong one? You might retransform E. coli with some of the original plasmid that looked positive, do minipreps on a few different transformants to confirm which are correct, and then use that DNA stock to proceed.
Thanks for your suggestion@Michael, I tried several times, and actually the beginning colony I selected is unique possibly because of the low efficiency. Some senior researchers said it possible that plasmid unstable because of eight similar promoter sequences used inside, though the homologolous recombination happened in E Coli or Agro, anotger suggestion would be select more stable competend cell, how do you think?
Also check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692276/
The E. coli strain EPI300 is a recA- strain so there should not be recombination taking place in E. coli. However that might not be true for Agro. I would still suggest you transform a very small amount of the original plasmid prep and then screen your transformants to see what they are like. You need a good starting source that you know is correct and pure before proceeding.
If you think there is instability in E.coli you could try using a different host strain like one of the Stbl strains.
Also, when you say the restriction profile changed, do you mean there is one additional or missing site, or is the profile really different and the sizes are different as well?
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