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Electrophoresis: sometimes mA=0


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#1 hianghao

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Posted 10 December 2010 - 02:00 AM

Hi,

I was conducting electrophoresis with TBE 0.5X, and found that the mA reading is 0. I've tried everything like changing the cables, generator and found that when i removed the buffer enough so that the buffer do not cover whole gel, the mA started to increase from 0 to some value. The last time i did electrophoresis, i remember that i cover the whole gel in buffer. What happened? I remember that i read from guideline which says the buffer must completely cover the gel to prevent melting...

I set my mA to be 300 but the max it goes is ~70, usually 30, and sometimes 10. IS this normal? Why can't it reach 300?

#2 bob1

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Posted 10 December 2010 - 07:22 PM

The electrolytes run out in the buffer, this is especially the case for lower ion concentration buffers such as 0.5x TBE, even more so if you have stored the buffer for a long time as some components precipitate. If the gel is still running (i.e. loading dye band still moving) when the current says 0, then it should be fine. You should cover the gel with buffer to prevent overheating.

#3 hianghao

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Posted 11 December 2010 - 04:41 AM

The electrolytes run out in the buffer, this is especially the case for lower ion concentration buffers such as 0.5x TBE, even more so if you have stored the buffer for a long time as some components precipitate. If the gel is still running (i.e. loading dye band still moving) when the current says 0, then it should be fine. You should cover the gel with buffer to prevent overheating.


I see. But the buffer in the tank was less than 1 week old... I'll try to replace the buffer next time. Thanks for the advise!

#4 HomeBrew

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Posted 11 December 2010 - 08:31 PM

The electrolytes are not exhausted by age, but by an electric field -- the positive ions go to the negative terminal, and vice versa. Once all the ions in the buffer have migrated to the poles, the buffer is exhausted and there is no circuit and thus no current. Try using 1X TBE or TAE and see if you maintain current. Either that, or your power supply or another component of the circuit is broken.




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