Huge DNA fragment cloning

[crotalus]

Hello everybody,

I work with reverse genetic systems for RNA viruses. It is very difficult to clone viral genes, since our viruses harbour GC rich sequences. Complete cDNAs are larger than 16 kb and are usually cloned in pBR322, because a Rob gene is needed for stability. These plasmids are exclusively proliferated in the old E. coli strain HB101 which accepts such ugly bugs, but the bacteria grow very tiny colonies. At the end of the day most of the plasmids still grow very poor , DNA is not easy to preparate and mutations are frequently generated . So my question:
Does anybody know more stable vectors for such large inserts?

Kind regards

crotalus

[mmorgan]

I have grown pretty large inserts of mammalian genomic DNA (20-30 kb) in pBlueScript-II. I know people don't usually recommend high copy plasmids for this kind of stuff but I always use them and I haven't had anything that I couldn't grow at all in pBSII yet.

I use the STBL2 cell line from invitrogen and grow at 30C, sometimes it is necessary to reduce the antibiotic concentration to get good plasmid yields.

Another option would be to use a BAC vector backbone which can hold much larger inserts (100-500 kb).

In terms of mutagenesis I don't typically sequence the entire insert (for my purposes this is not necessary), but the parts that I have sequenced (say approximately 30%) have not had mutations.

[crotalus]

Thank you for your suggestions mmorgan!

I tryed several standard plasmids (like pBSII, which is helpfull for the cloning of small RT fragments of my virus). But I was never able to clone more than a single GC rich gene of my virus in such high copy plasmids. Currently, I grow my bacteria at RT with reduced (1/4) ampicillin concentration for 3 days. A single mutation is often fatal for virus replication, so it is a hell of a work to clone a new virus strain (more than one year!). I really like plasmids and am not willing to start BAC work at the moment. I hope that someone from this community has solved the plasmid stability problem.