Short version: My plasmid contained insert but later after a second transformation into a second cell line the insert vanished and does not show up on PCR or sequencing
Long version: I used ligation to add my insert (1 kbp) to my plasmid (5 kbp). I then transformed JM109 cells and plated overnight. Afterwards I used A gotaq pcr kit to test the colonies directly for the presence of my insert. All 8 of the colonies tested had the insert. I then grew a JM109 culture overnight and harvested the plasmid using a maxiprep kit. I then used the harvested plasmid to transform BL21 cells for protein expression. However the plasmid sequenced after the maxi prep step appears to no longer contain the insert. The sequence corresponds only to unaltered plasmid. Additionally no PCR products can be created from the plasmid, using the primers used to create the insert.
How is this possible? My best guess is that I transformed the JM109 cells with a mixture of plasmid that contained the insert and pure unaltered plasmid (that must self ligated). Over time the unaltered plasmid has come to dominate and the insert containing plasmid is now too weak to detect?
however each colony is supposed to be clones of a single e. coli cell. If they once possed plasmid with the insert should they not all still have it present?
Hi Gary,
it is most likely that one way or the other you observe experimental an artefact. Most typically false-positive, false negative and/or contamination. It is best to start again, make sure all your reagents, plasmids etc. and are what you think they are. Only after you are really sure that your insert is indeed deleted you should start thinking about this. some inserts are toxic to the bacteria (especially in expression strains) and thus there is selection pressure to eliminate it or clones recieving a "cloning artefact" (e.g. vector religation) are favoured. In these cases it can help to ensure to repress expression (e.g. 2% glucose), keep incubation times as short as possible, and maybe reduce temperature to 30°C. If nothing helps you may look for very tightly repressed systems, or even use phage infection for induction. Also, check the genotype of your bacterial strains (https://openwetware.org/wiki/E._coli_genotypes)! You'll notice that a "cloning strain" is recA (https://en.wikipedia.org/wiki/RecA) negative but many "expression strains" are not.
Best regards, Markus
It is a bit too good for your first PCR screening, all 8 were correct. One of the possibility is that all 8 colonies were not right, but your PCR picked something else.
I have actually only recently begun the practice of testing the colonies. Before I have always assumed the antiobiotic has succesfully screend for transformation adn would just randomly select a colon.
I have never before actually encounteded a false positive.
Hi Gary,
it is most likely that one way or the other you observe experimental an artefact. Most typically false-positive, false negative and/or contamination. It is best to start again, make sure all your reagents, plasmids etc. and are what you think they are. Only after you are really sure that your insert is indeed deleted you should start thinking about this. some inserts are toxic to the bacteria (especially in expression strains) and thus there is selection pressure to eliminate it or clones recieving a "cloning artefact" (e.g. vector religation) are favoured. In these cases it can help to ensure to repress expression (e.g. 2% glucose), keep incubation times as short as possible, and maybe reduce temperature to 30°C. If nothing helps you may look for very tightly repressed systems, or even use phage infection for induction. Also, check the genotype of your bacterial strains (https://openwetware.org/wiki/E._coli_genotypes)! You'll notice that a "cloning strain" is recA (https://en.wikipedia.org/wiki/RecA) negative but many "expression strains" are not.
Best regards, Markus
Your observation can be explained by double-transformation artefact, when one cell takes two plasmids. It is underappreciated, because in most applications one does not notice them. However, the frequency of such events is not that low, and two plasmids within a cell can survive for many generations. check the link on the topic:
https://www.ncbi.nlm.nih.gov/pubmed/17575283
In your case it's very likely that the cells do not like the insert and select against it. Therefore I would go for a tighter repression vector.
Many years ago Herman Bujard showed that there was active deletion of inserts when which transcription rates were induced, not just counter selection of plasmid hybrids. Although unlikely one can keep the instability in prokaryotes of palindromic (two-fold inverted symmetry) DNA in mind. see: Collins J. Instability of palindromic DNA in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):409–416. [PubMed]
- Collins J, Volckaert G, Nevers P. Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli. Gene. 1982 Jul-Aug;19(1):139–146. [PubMed]
Of course one should always do each experiment three times at least to be sure and more if it requires some statistical analysis of root mean variance. This is a basic rule in science. Trying to make short cuts is just wasting everyone’s time.
When you PCR, use one plasmid-specific primer and one gene-specific primer so that only a ligated product will give you a positive result. In my experience, using only gene-specific primers can result in false positives, presumably due to unligated products picked up from the plate when you pick up the colony.
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