Open debate: agarose inhibition :-/

ChenHA hachen at bc.ic.ac.uk
Thu Jun 10 08:30:18 EST 1999



On 9 Jun 1999, moez torki wrote:

> Hi Molbiolers,
> 
> I often heard that gel purified fragments can be recalcitrant to subsequent
> enzymatic treatments (eg. phosphorylation, ligation, etc..). Although I
> never had problem before, now I am experiencing serious problems in my new
> lab :-( That is I cannot perform even a rudimentary subcloning of my
> TAE-agarose/Geneclean purified fragments. I could get rid of that problem
> by phenol/chloroform/EtOHppt. I could see a whitish pellet and a white
> interface after phenol extraction that I may guess being agarose ? or
> silica ?

Always use low melting point agarose, then you would not have any
problem.  I believe the contaminant in the normal agarose which affect
ligation is sulphated agarose.  

Moving from one lab to another often bring surprising problems.  I had
some trouble with growing up bugs in minimal media in my new lab using
my trusty recipe that have always worked before.  I now find out what
the problem is - the water here is filthy.  So now I use water from
another lab and there's no more problem.  

> Has anyone bear such troubles ? and is there really significant differences
> in agarose quality (depending on the supplier/price). Indeed I can carry on
> with the phenol extraction but it doesn't really make sense since I am
> using kits to get results faster...
> Thanks for any comment.
> 
> Moez Torki





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