cloning in M13

Alexander Kraev kraev at bc.biol.ethz.ch
Sun Sep 1 10:33:58 EST 1996


In article <506i95$up9 at venus.plain.co.nz>, tonyr at chch.planet.org.nz (Tony
Raizis) wrote:

> Has anyone out there had experience with cloning large fragments i.e.
> greater than 1kb in M13mp18? The literature supposedly states that it's
> no problem if you use a competent cell strain which is recA- e.g. JM109.
> I would feel more comfortable if someone with hands on experience could
> verify this and perhaps give us a few useful hints on the matter.
> 

The size of the plaques gets progressively smaller with larger inserts, which
is one of the main reasons why these clones are not found at all, or claimed
to be "unstable", because any neigbouring wildtype plaque contamination is
very likely to outgrow the main one in liquid culture. The feature of reducing
the plaque size is also sequence specific and handling of such sequences
is not easier, if one first clones into a phagemid and then packages the single
strand ( but at least you know you have the clone). Essential for success in
cloning large fragments is the use of fresh agar plates and drying only 10 min
after the soft agar has set, as well as rigorous repurification of the clone
by re-streaking.

I hope this helps,



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