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.
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