Dear all,
I am planning to clone a gene in pET24a(+) Series vector but before going on for cloning i tried to sequence pEt24a(+) null vector from our lab repositiory, but in sequencing results we are getting mutations in promoter region. Is it normal that vectors get mutated ?
Dear Vishal,
I am not surprised to hear that you have identified mutations in the prompter region of pET24a. I would suggest a simple explanation : unwanted transcriptional activity from this site has a negative (fitness) impact on the host cell - plasmid system, and over time, perhaps as colonies are maintained on plates (rather than stored at -80°C) mutations are selected for which reduce promoter activity. I would expect to see similar examples of mutations in other protein expression plasmids, especially when the strains are maintained on plates with incomplete repression, or in the wrong host back ground.
You should try to find another source of the plasmid for your experimental work, and maintain it in an appropriate E. coli host and stored at -80°C until samples are required.
Regards, Andrew.
Dear Vishal,
I am not surprised to hear that you have identified mutations in the prompter region of pET24a. I would suggest a simple explanation : unwanted transcriptional activity from this site has a negative (fitness) impact on the host cell - plasmid system, and over time, perhaps as colonies are maintained on plates (rather than stored at -80°C) mutations are selected for which reduce promoter activity. I would expect to see similar examples of mutations in other protein expression plasmids, especially when the strains are maintained on plates with incomplete repression, or in the wrong host back ground.
You should try to find another source of the plasmid for your experimental work, and maintain it in an appropriate E. coli host and stored at -80°C until samples are required.
Regards, Andrew.
I agree with Andrew Spiers. Plasmid stock E. coli strains maintained on plates will almost certainly lead to the accumulation of mutations in the plasmid.
Are any of your plasmid stocks in E. coli maintained at -80oC? If not, you should start doing that! Be sure to add sterile glycerol to a final concentration of 15% before freezing. This stabilizes the cell envelope.
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