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Agarose gel electrophoresis problems


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#1 greenhill

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Posted 24 April 2009 - 03:39 AM

Please help! :unsure: I am having a problem running my agarose gels. The ladders always end up looking very distorted. The bands are smeared towrdas the top end, but fairly well defined at the bottom end. The middle looks like all the bands have landed on top of each other. I have tried different voltages from 80-120V and different buffers (TAE and TBE), but to know avail. In the attached image, I tried a series of different ladders to see if it was the particular ladder I was using. Does anyone know what the problem could be?

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  • 204506963_2009_04_21_15hr_17min.jpg


#2 Kami23

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Posted 24 April 2009 - 04:15 AM

Please help! :unsure: I am having a problem running my agarose gels. The ladders always end up looking very distorted. The bands are smeared towrdas the top end, but fairly well defined at the bottom end. The middle looks like all the bands have landed on top of each other. I have tried different voltages from 80-120V and different buffers (TAE and TBE), but to know avail. In the attached image, I tried a series of different ladders to see if it was the particular ladder I was using. Does anyone know what the problem could be?


it looks to me like your ladder is degraded.. how old is it? also do you change tips every time you dip in (EtBr will degrade the DNA)? Do you aliquot it first (this protects against the effects of freeze-thaw)?

#3 HomeBrew

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Posted 24 April 2009 - 04:49 AM

If it's not the buffer, and it's not the ladder, it's either the gel or the equipment.

Are you sure you're casting the gels in the buffer you're running them in, and not in water? Are you sure the buffer is the right strength?

Do you have another power supply you can try?

Is the power supply set to constant voltage or constant current?

Do you have another gel electrophoresis chamber you can try?

#4 mastermi

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Posted 24 April 2009 - 07:54 AM

Your whole gel looks irregularly stained, like the agarose didn't melt completely. That could be due to either the agarose or the buffer your cooking it in...

#5 Astilius

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Posted 24 April 2009 - 08:39 AM

As mastermi says it looks irregularly stained. Has it been allowed to set properly and not been moved around. It looks like ripple lines at the bottom there.
It might be a density problem related to this or the agarose not being melted properly.
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#6 mdfenko

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Posted 24 April 2009 - 10:35 AM

is there salt left in your samples?
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#7 HomeBrew

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Posted 24 April 2009 - 09:00 PM

From the original post, it seemed like greenhill has had this problem with a number of gels, and the run shown is of MW ladders only. So, a persistent problem like that makes me think it's a persistent buffer or gel ingredient preparation problem or an equipment problem, and not a sample problem. It also seems like it's probably not due to incomplete melting or hardening of the agarose, as I could see this happening on occasion, but not on multiple consecutive gels (though this problem would likely manifest itself in this way).

#8 Niraj

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Posted 25 April 2009 - 03:38 PM

Hi,
Please stirr after every few min of boiling youer agarose and they will disappear.
Best of luck.

Edited by nirajm, 25 April 2009 - 03:38 PM.





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