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size limit of plasmid origins

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#1 evolver



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Posted 08 April 2009 - 11:09 PM

Hi all,

I am interested in prokaryotic (E. coli) origins of replication. Does anyone know of a reference giving the theoretical or empirical size limit of DNA that can be stably maintained from different origins of replication?

In particular, I am interested in the P15A origin. I have found a reference where it could support a 10.6kb plasmid. But, I would like to use this origin and a chloramphenicol resistance gene to maintain a 16kb piece of polycistronic DNA, final size of the plasmid would be about 19kb.

Can anyone say if this is in principle possible? or if I should go straight for a cosmid origin, like oriV which we know can support 40+kb?

Thanks a bundle!

#2 phage434



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Posted 09 April 2009 - 04:04 AM

This will probably work, but I don't have a reference. DNA of this size is more difficult to transform, and is fragile in solution. Avoid vigorous pipetting and vortexing. Why not move to a BAC plasmid origin?

#3 evolver



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Posted 09 April 2009 - 08:13 AM

Why not move to a BAC plasmid origin?

I think a BAC origin is probably the best idea. But we don't have any in our collection at the moment (although I can probably get one from somewhere...), AND it just so happens that I have a plasmid with the P15A ori and Chloramphenicol resistance conveniently next to each other. So I can easily PCR it and incorporate some regions for recombination with the 16kb insert.

I think I will trust your and my gut feeling that this will probably work and give it a shot. It is less than a week to have an indication of success so not too much is lost in the case of failure...

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