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Need ideas for band seperation

Need ideas for band seperation - Agarose Gel Electrophoresis Forum

Need ideas for band seperation - Agarose Gel Electrophoresis Forum. A forum to discuss electrophoresis staining, troubleshooting, and development of films for DNA and RNA gels.


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  #1  
Old 06-01-2010, 09:52 PM
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Default Need ideas for band seperation



Hi,

I am trying to sequence a gene that appears to have a duplication and the duplication is 20-70 bp less than the original. I've tried three genus specific primer sets and observe the double band every time, so I know it isn't contamination or mispriming.

Attached is an image of the PCR product visualized on a 3% agarose gel after 45 minutes. The bands are too close together to extract for sequencing. I ran the gel again for 150 minutes but the bands became very weak and still were'nt seperated enough to confidently remove them.

Is there any way to get these bands separated? Any suggestions are welcome and appreciated.
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Old 06-04-2010, 04:05 PM
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Default Re: Need ideas for band seperation

Have you tried high resolution agarose? If doesn't work, polyacrylamide should do.

Hope it helps ^^!
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Old 06-08-2010, 10:18 AM
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Default Re: Need ideas for band seperation

We separate bands of 409bp and 440 bp with no problems. We run a 1,6 % agarose gel at 120V for 35 min. So maybe you could try to run a gel with less % agarose.
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