large (>60 kb) multicopy plasmid replicons

Peter Wang plw at mrc-lmb.cam.ac.uk
Sun Nov 26 12:04:00 EST 1995


aiyar at biocserver.BIOC.CWRU.Edu (Ashok Aiyar) wrote:
>I am looking for a source (or references to) E. coli plasmid
>replicons that support the stable replication of large plasmids
>in the range of 5 - 20 copies per cell (or greater).
>
>We are currently using an F episome that is around 70 kb to
>carry a large portion of Epstein Barr Virus in it.  This 
>replicates at about 1 copy per cell, which makes DNA isolation
>for cloning and transfection purposes rather difficult.
>...

Ashok:
You might try the temperature-sensitive R1 replicon:  at 30 C it has a 
copy number of one, but at 42 C it loses copy-number control and 
replicates in a runaway fashion - after 1-2 hr it is reported that 50% 
of the DNA is plasmid DNA (>1000 copies/cell).  No, I haven't used it 
myself - in fact, if you find the plasmid, I'd be interested in getting 
some.  The reference I have is:
     Larsen JEL et al., Gene 28 (1984):45-54
Cheers,
- Peter







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