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-   -   Frowning Gels (http://www.molecularstation.com/forum/agarose-gel-electrophoresis-forum/83509-frowning-gels.html)

DacotahM 09-07-2011 07:57 PM

Frowning Gels
 
I left the lab for a few weeks and now our gels aren't behaving properly.

The loading dye (bromophenol blue) doesn't migrate into the gel easily and severely distorts the PCR product behind it. The dye bands form a large frowning arch when placed in adjacent wells. Individually the outside edges of each band runs much farther and the center of the band drags behind creating a frowning (

Things I\'ve already tried:

-make new TBE
-scrub out the gel trays
-make new blue loading dye

I also tried Gotaq Green buffer which contains a loading dye diluted with PCR water, and this also frowns... The ladder runs fine.

The loading dye will sit in the wells much longer than normal- for the first 20 minutes or so it doesn\'t seem to leave the well. Then it begins to tug out at the edges.

The agarose is 1%.

obama 09-07-2011 09:25 PM

Re: Frowning Gels
 
Bit strange Problem. only reason might be high salt in your sample.

With Regards
Uday Kumar
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DacotahM 09-08-2011 02:04 PM

Re: Frowning Gels
 
All I'm running is loading dye and water.

obama 09-08-2011 02:32 PM

Re: Frowning Gels
 
Are you diluting enough before you running it

DacotahM 09-08-2011 03:43 PM

Re: Frowning Gels
 
Yes, to the standard dilution we've always used when running samples. I also tried 2 commercial loading dyes. I've also done serial dilutions for all 3.

obama 09-09-2011 11:03 AM

Re: Frowning Gels
 
And how much volt are you using. and chamber size. Some time this also affect the migration.

DacotahM 09-09-2011 03:48 PM

Re: Frowning Gels
 
I have tried absolutely everything except replacing the EDTA and Boric acid.

butters 10-01-2011 03:33 AM

Re: Frowning Gels
 
Is there by any chance the agarose you use is different? such as not LOEE?


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