plasmid copy number - experiments on how to compare the copy numbers of different copy-numbe (Jun/01/2008 )

Dear colleagues,

I changed the ori of the plasmids to get putative high copy-number plasmids, but now I am not confirmed that the ori-changed plasmids yeald more than that intact-ori plasmids in the same E. coli strain.

So I need design an experiment to test the plasmid copy number to check which one is high copy-number plasmid.

Anyone has the experience can tell me, thanks.


Jackie

From Changchun Institute of Applied Chemistry, CAS
Changchun
P.R. of China

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One of the approaches you can use is relative real-time PCR. You design two pairs of primers, one for a certain fragment of downstream of ori, another one for an endogenous gene (as for normalization), then run real-time PCR. Good luck.

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Cuantitation for real time PCR would be good to know the copy number, but I think that comparing amount plasmid DNA extracted by normal minipreps from same amount of culture (same O.D. or wet weight biomass) would be enough to know which one has the highest copy number.

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I like this approach, a lot simpler.

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