subcloning of rearrangement-prone DNA

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Sat Nov 1 05:14:46 EST 1997


Hongyan --
    I just cloned a 3.2kb K+ channel which is 65% GC overall with stretches
of 80% GC, and I got all sorts of rearrangements at differents times.  I
did not like working with SURE or TOP10F' because of lack of colonies
(although quite competent).  One thing I found helpful was getting out of
Bluescript.  I had a lot more luck with pGEM (3zf-), and I am told that
pUC18/19 is equally reliable.  I ended up in XL1 blue MRF', which does
sometimes rearrange, but that is better than no colonies ever.
    Harry

Hongyan Liu wrote:

> Everyone,
>
> I have had problems with subcloning one piece of mammalian DNA . With
> DH5alpha cells, I got apparently rearrangement of that DNA when ligated
> to pBluescript. Now I tried Sure cells bought from Stratagene but have
> not got any colonies (almost no background blue colonies as well).
> However, pUC18 control plasmid did give some colonies.
>
> Any ideas what I can try next?
>
> Thanks.
>
> HL






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