This may be a simple and old question. People usually pick single colony of E coli to purify plasmid, which is considered as identical plasmid (a single clone). I just wonder whether this is always true, especially when generating point mutations. Is there any published studies to address this question?
It is usually assumed that one colony of E. coli has only one type of plasmid. But this is certainly not always true. I have literally done 1000s of blue/white screens and have certainly seen my fair share of sectored colonies, which indicate plasmid instability.
The fact that each colony of E. coli could have multiple plasmids is the reason why it is always a good idea to run a sample of your plasmid prep on a gel before transformation...never hurts to be overly cautious.
About your other question regarding literature...I don't know of any off hand that relates to E. coli specifically. However, there is a classic example of what you are concerned about in the organism Halobacterium sp. NRC-1. Almost every time you plate NRC-1 you get pink and red colonies. This is due to insertions in one of the plasmids, which interrupts the gas vesicle genes and causes the color change. A good place to start reading on this is here...http://link.springer.com/article/10.1007/BF01955149
Selection of single colony is generally considered as clone, but it need not be correct always. Copy number of plasmids differ from plasmid to plasmid and some plasmids are compatible and other are not.
If you have mix of mutant plasmids used for transformation, a single cell may get transformed with more than one plasmid. Over the time (after some number of cell divisions) one or the other mutant persist and others will vanish because of genetic drift (plasmid incompatibility).
If you are expecting mixed plasmids (point mutants) I suggest to select number of colonies, grow them in broth culture overnight, spread on a plate and select a few colonies and repeat the selection for at least three times to get fairly identical clones.
yes it could. but if you have frequency in culture its more trust able than single colony. but you have chance to get your sequence target.
It is usually assumed that one colony of E. coli has only one type of plasmid. But this is certainly not always true. I have literally done 1000s of blue/white screens and have certainly seen my fair share of sectored colonies, which indicate plasmid instability.
The fact that each colony of E. coli could have multiple plasmids is the reason why it is always a good idea to run a sample of your plasmid prep on a gel before transformation...never hurts to be overly cautious.
About your other question regarding literature...I don't know of any off hand that relates to E. coli specifically. However, there is a classic example of what you are concerned about in the organism Halobacterium sp. NRC-1. Almost every time you plate NRC-1 you get pink and red colonies. This is due to insertions in one of the plasmids, which interrupts the gas vesicle genes and causes the color change. A good place to start reading on this is here...http://link.springer.com/article/10.1007/BF01955149
We just published a paper to address this question.
http://www.pubraw.com/journal/view.html?id=109
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