Separating PCR products on agarose gel with similar bp length

umwong25 from cc.umanitoba.ca via methods%40net.bio.net (by umwong25 from cc.umanitoba.ca)
Fri Dec 4 20:13:23 EST 2009


Hi Peter,

Thanks for the suggestion but due to recent events the lab I work at  
does not have the capabilty to do cloning. I also want to see if there  
are any other similar sized fragments (possible splice variants) in  
the amplified region of the gene.

At Fri, 04 Dec 2009 22:24:55 +0000, Peter Ellis wrote:

> umwong25 from cc.umanitoba.ca wrote:
>> 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?
>
> Would it be simpler to clone the mixture and then just sequence half a
> dozen clones until you get the right one?  Or is the band you want much
> weaker than the others?
>
> Peter
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