After a serial passage of plasmid in E coli, I extracted the plasmid from bacteria colonies and sequenced to identify mutations. The peaks were quite nice with almost no background noise but what puzzled me was that fact that some peaks were double and some triple comprising different nucleotides. It appears the plasmid had some mutations in the course of passaging and the mutants bacteria grew and together formed the colonies. I find it difficult to interpret and I need your help.
The multiplicity could have occurred at several different levels:
1) multiple bacterial colonies could have formed "on top of each other".
2) multiple plasmids could have been taken up be a single bacteria.
3) the insert could have integrated multiple times (e.g. concatemerized).
More experimental detail are needed to evaluate these (and possibly additional) different options, but I will leave that to you ;-)
Based on your experimental setup (several passages of the plasmid in E.coli), I would guess that the individual plasmid copies within each cell of colony might carry differnt mutations. That should be a rare occurrence unless you apply some kind of selective pressure...
How many passages did you perform?
I performed 10 passages in Luria Broth + ampicillin because the plasmid has an ampicillin resistant gene as a selectable marker.
Sounds unsuspicious. But why did you do the passages in the first place?
Anyway, if you then plated the bacteria and picked single colonies for plasmid isolation and sequencing, this indicates that you have indeed mutations ind the different copies of the plasmid inside the cells. If you want to test this, you might do one or two rounds of picking and streaking a single colony. Of course, this "polyploidic" trait might not be stable.
HI.. i would also do as Christian Rückert says. Pick up a single colony and streak it out on a plate and pick colonies from that and try to sequence it.
Now I am a bit baffled. If you want to determine plasmid stability, why do you add the antibiotic which selects for the presence of the plasmid?
Or did you want to test the stability of your insert? In that case, your results point to some kind of pressure to accrue mutations...
Hi Christian,
We have two plasmids containing the same insert and will want to ascertain the stability of the insert in the respective plasmids. We simultaneously passsaged both plasmids in E coli and BHK21 cells.........................Clones, plaques and viral supernatant were sequenced to identify mutations (if any) in the insert.
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