DNA isolation from agarose gels

Eric R. Hugo hugo at nku.edu
Tue Mar 9 07:20:18 EST 1999


In article <KTZD2.351$6D3.7799136 at newsie.cais.net>, "Sean Fitzsimmons" <fitzsp at cais.net> wrote:
>What is the simplest and most efficient method for recovering restriction
>fragments from agarose gels.  I am trying to recover PCR product whose ends
>have been digested with restriction enzymes from HMP agarose.  The gels have
>loads of product, but recovery of approximately 5% seems to be the norm.
>I've tried Wizard PCR preps, Quiex II, and electroelution, all with very
>dissappointing yields.  Any suggestions of simple, high-yield protocols?
>
>Thanks in advance.
>
>Sean Fitzsimmons
>
>
One thing I have found is that staining the gel with Methylene
blue instead of ethidium bromide greatly improves efficiency
of subsequent cloning of band isolated fragments (50x increase).
 I have had much success in combining this staining technique
with GeneClean/glassmilk protocols.  It also is important to use
TAE rather than TBE running buffer.  Hope this helps.
Eric


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