During DNA transformation experiments, we sometimes see a few false positive transformants ---- bacterial colonies that carry no any insert plasmids or DNA fragments but can still grow on antibiotics plates. What do you think are the causes of this phenomena? Assuming that antibiotics in agar plates are in their normally effective concentration range.What could happen to the bacteria? How likely is it because of spontaneous mutations?
Hi there,
What you call false positive transformant is actually a cell containing an empty plasmid (ie a plasmid without the expected insert). The presence of empty plasmid in your transformation mix is the consequence of the unefficient digest for opening the starting vector and/or the self ligation of the plasmid. Therefore the frequency of clones containing the empty vector is highly dependent on the digest efficiency and the ligation efficiency. If these both steps are optimized you shouldn't have a lot of non positive transformants.
Hi there,
What you call false positive transformant is actually a cell containing an empty plasmid (ie a plasmid without the expected insert). The presence of empty plasmid in your transformation mix is the consequence of the unefficient digest for opening the starting vector and/or the self ligation of the plasmid. Therefore the frequency of clones containing the empty vector is highly dependent on the digest efficiency and the ligation efficiency. If these both steps are optimized you shouldn't have a lot of non positive transformants.
Thank you, Dominique. But how about if I use un-replicated plasmids for a gene knockout? Those colonies are not the real knockouts but they can still grow. What might happen to the bacterial cells?
Hello,
Like Dominique said, the problem of false positives is mostly due to an inefficient digestion/ligation. Whenever you use a template plasmid (which carries the antibiotics resistance), there is almost never the chance for full digestion of the template, leading to "empty" parental plasmids.
Another very simple thought is the age of your antibiotics plates and the time of incubation. This could also lead to false positives.
But I guess it would be helpful if you explain which method you perform. And how do you suppose that they don't "carry" any DNA fragment?
Hi again,
Right so you are using plasmid for genomic insertion and gene disruption . The false positives you are talking about result from plasmid insertion elsewhere in the chromosome. If the frequency is high it means that insertion at the right place is problematic (lethality by inactivation of the gene targeted?)...
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