DH5alphas are growing slowly.

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Aug 7 03:44:40 EST 1998


Darren Boehning wrote:
> 
> I have noted the same problem lately in my own cloning projects, but with
> high copy number/very common vectors (i.e. pcDNA3.1).  I just happened to
> leave the plates an extra 24hours and found the same surprise.  I screened
> all of the "48hr" colonies, and not one of them was positive. 
> I have
> since finished several of my projects, and they were all successfully
> completed from "24hr" colonies.

That is my experience as well. It is not worth to let the plates in the
incubator until colonies appear: When colonies aren´t evident after some 18h,
discard your plates and re-try cloning.
In most cases I have seen, you cannot prepare plasmid DNA from colonies that
need 48h to become evident. Together with the facts that a) you don´t see
these colonies on a cells-only control plate, and b) the effect is most
evident when cloning with lots of vector DNA, I have concluded that the effect
probably comes from integration events of (linear) vector into the bacterial
genome. In some cases the amp resistence will then be expressed, and you get
amp resistant colonies that grow slowly but do not contain a plasmid. Well, I
said PROBABLY, because I have no hard data on that.

Frank


>  I have also noted that there are large
> variations in colony size after 24hours. Has anyone noted whether colony
> size has anything to do with obtaining a positive clone? (I am personally
> biased towards larger ones!).

When there are LARGE variations in colony size, you might have a mixture of
two strains. Streak to single colonies (on non-selective plates) to "purify" a
defined bacterium. SOME variation in colony size is quite common (especially
when using expression vectors), and I usually take the smaller colonies with
good success.

Frank



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