Open debate: agarose inhibition :-/
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...
Institut de recherche en Biologie Vegetale
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