Inserting a large DNA fragment into a large plasmid

CSC un691cs at genius.embnet.dkfz-heidelberg.de
Fri Sep 17 07:58:00 EST 1999


On Fri, 17 Sep 1999, Mike Cooley wrote:

> "Shinohara, Mari" wrote:
 
> > Hi All,

> > I have a plasmid which has a 10-kb insert in pBluscript and still have to
> > put either 2-kb or 6-kb flagment elsewhere at the middle of the insert.
> > Since the total size of a resulting plasmid would get either 15-kb or 19-kb,
> > it may be almost impossible to get it transformed into E. coli after
> > ligation.

> > So I am now thinking to use a phage to clone the resulting big fragment, but
> > I have never used phage for such a cloning purpose.  If you have any
> > suggestions or other strategies for this type of cloning, anything would be
> > greatly appreciated.

> > Mari Shinohara

> The pBluscript  vector does have an upper limit, but in your case I don't think
> you will exceed it. I have pBluscript plasmids that size before.

Still, the cloning efficieny may well go down, as you try to put a big
insert into an already big vector. I would try it anyway. Try to make a
targeted cloning if possible, sticky sites on both sides. I wouldn't use
blunt end cloning. Gelpurify the digested plasmid (may be tough at this
size, run a 0.8% gel). Use varying amounts of plasmid (10ng-500ng) and 100
ng of insert). Perhaps you could try colony hybridization or colony PCR to
identify the proper clone ?


Kind regards,

Clemens Suter-Crazzolara, PhD

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