Separating PCR products on agarose gel with similar bp length

Michael Mattocks via methods%40net.bio.net (by mike.mattocks from utoronto.ca)
Sat Dec 5 13:22:56 EST 2009


A 10% non-denaturing polyacrylamide gel will probably do the trick. If 
you're familiar with SDS-PAGE this isn't much different, leave out the 
SDS and run in 1x TBE (or .5x TBE if you prefer). Everything else in 
terms of loading dye, markers and so on you can keep the same. It's a 
bit easier to overload the gel so try a modest amount of product first. 
You can extract the band for sequencing with an ammonium acetate 
crush-and-soak protocol.

Mike


> Message: 1
> Date: Fri, 04 Dec 2009 15:17:08 -0600
> From: umwong25 from cc.umanitoba.ca
> Subject: Separating PCR products on agarose gel with similar bp length
> To: methods from magpie.bio.indiana.edu
> Message-ID: <20091204151708.jz3gv073s0kg8wko from webware.cc.umanitoba.ca>
> Content-Type: text/plain;	charset=ISO-8859-1;	DelSp="Yes";
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> 
> Hi,
> 
> I am trying to separate 745bp, 646bp, and 655bp fragments on an  
> agarose gel. I've separated the 745bp fragment but the other two  
> fragments are not separating on a 2.5% agarose gel, running at 55V for  
> 3.5 hours. I also want a crisp band because I am planning to cut these  
> fragments out and extract the DNA for sequencing. Any suggestions?



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