DNA elution from agarose-gel

DK dk at no.email.thankstospam.net
Fri May 13 08:19:38 EST 2005


In article <v1m881973hlrkfbdn4c97sth6g5b4esna4 at 4ax.com>, Dirk Müller <Kraweel at web.de> wrote:
>We have a severe problem with eluting restircted DNA (10-15kb) from
>agarose gel. 

For a 12.5 kb cut plasmid that I needed to purify, I used Qiagen's
Gel Elution kit (pink columns). Recovery was over 50% when eluted
with the hot (~70C) 2 mM tris pH >8.0. I see no reason why it should
be any different for you.  

>We have already testet QiaexII, Genomic purification kit
>from Promega, Gel elution colums from Microcon, and electroelution but
>don't get more than 1-10 % of the initial DNA which is not enough for
>downstream methods. Using electroelution we can elute the complete DNA
>into the membrane(DEAE or hybond N/N+) but we can not elute the DNA
>from the membrane in the elution buffer (15 min at 65°C in 2 M NaCl,
>10 mM Tris/HCl pH 7 (pH 7-8), 1 mM EDTA). 

I'd say for near quantitative recovery you need to use electroelution.
Don't use any binding membrane - just place gel fragment into 
dialysis bag and run for long enough. 

DK




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