transformation inhibition due to gel extraction?

David Winterbourne sghk100 at sghms.ac.uk
Tue Nov 18 04:44:10 EST 1997



Nico Dantuma wrote:

> Hi,
>
> The last few weeks I have been struggling with some persistent cloning
> problems.  All the obvious steps (digestions, dephosphorylation and
> ligation) were checked and no problems were observed. Yet, even after
> transforming bacteria with ligation in which ligated products were clearly
> visible on an agarose gel, no transformants were obtained ....

> Does this problem sound familiar to one of you and more important what can
> I do to overcome this problem?

Yes, sounds familiar.

Try different batches of agarose. High purity agarose is tested for
interference with enzymatic reactions (ligation, restriction etc.), but as far
as I can tell, is not tested for inhibition of bacterial transformation.

My experience was similar - ligation products were clearly visible on the gel,
but I got no transformants. There was something toxic extracted from the
agarose coming through a Wizard clean up (it inhibited transformation by a
standard plasmid prep). I overcame the problem by changing to a high grade
low-melt agarose and performing a phenol chloroform step before the Wizard
clean up (belt and braces approach).


--
David Winterbourne
Department of Surgery
St George's Hospital Medical School, London SW17 0RE, England
Tel: 0181-725-5581   Fax: 0181-725-3594





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