Large plasmid cloning

chmc chmc at chmc.org
Tue Apr 13 17:10:52 EST 2004


"ChenHA" <hzhen at freeuk.com> wrote in message
news:4070BA9A.FF8D91B5 at freeuk.com...
>
>
> b8703126 at sinamail.com wrote:
> >
> > Hi,
> >
> > The vector was linearized and dephosphorylated by CIP, and purified by
gel-extraction.
>
> Can't really say much without actually seeing how you do things, but
> one things I find that can affect ligation efficiency is the
> dephosphorylation step by CIP.  I tend to avoid using it, and if you
> really need to (in your case if you are using only a single
> restriction site), use only the minimal amount recommended.
> Personally I would simply try, if possible, ligating using two
> restriction sites and leave out the CIP step.
>
>
I agree.  You get less of everything when you CIP.

> > The insert was also purified by gel-extraction.
>
> Always a good idea to gel-purify, but a faulty transilluminator can
> affect ligation cloning efficiency, although if you are using one that
> hasn't been any problem before this is unlikely to be a cause.
>

I've also had trouble with DNA from some brands of agarose.  Low Melt seems
to work well for me, but maybe that's just superstition.






More information about the Methods mailing list