Open debate: agarose inhibition :-/

Caron Sebastien carons at MAGELLAN.UMONTREAL.CA
Thu Jun 10 07:53:54 EST 1999


On 9 Jun 1999, moez torki wrote:

> Date: 9 Jun 1999 19:30:10 -0700
> From: moez torki <torki at NOCS.TSUKUBA-NOC.AFFRC.GO.JP>
> To: methods at net.bio.net
> Subject: Open debate: agarose inhibition :-/
> 
> 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 ?
> 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
> 
> _________________________________________
> Moez Torki, Ph.D.
> Plant Biotechnology Institute,
> Ibaraki Agricultural Center, 3165-1 Ago,
> Iwama, Nishi-Ibaraki 319-0292, Japan
> 
> Tel:   +81-299-45-8330
> Fax:   +81-299-45-8351
> Email: torki at nocs.tsukuba-noc.affrc.go.jp
> _________________________________________
> The World : once thought to be flat, then proved to be round. Now it's
> quite definitely Web shaped....
> 
> 
> 
We have had the same problem in our lab while constructing a
transformation vector and the only answer we found was to skip the gel
purification step and directly do a shotgun ligation. Of course, you will
have to screen for the good ligation product but at least it works this
way. I suppose that agarose quality would definitely be important but
then, we had top quality agarose and it didn't help us any better...

Sebastien Caron
Institut de recherche en Biologie Vegetale
Montreal, Canada




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