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Electrophoresis problems


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6 replies to this topic

#1 Ameya P

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Posted 08 October 2010 - 04:42 AM

Hi all,

We use 0.5X TBE buffer and 150V for electrophoresis in our lab. Although, I regularly change the buffer in the tank, it is often that the buffer heats up quite quickly during electrophoresis. (I know that buffer acts as a coolant as well

I also found out that we add too much EDTA when preparing the stock solution. (almost 42gms instead of the suggested 9gms to a litre).

So I am wondering, if this is the reason why the buffer heats up? If not, what should be done to reduce the heating issue?

Aditional info: I am the only one using the tank and on an average run a gel a day and change the buffer every fortnight.


Thanks
:)

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#2 mdfenko

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Posted 08 October 2010 - 07:14 AM

prepare fresh buffer with the correct amount of all components.

excess of any component will cause heating (higher ionic strength will cause higher current flow and higher wattage).
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#3 LabLackey

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Posted 08 October 2010 - 07:17 AM

Sounds to me like adding excessive EDTA could lead to high buffer heating. The more salt or ions you have in solution the better it will conduct current (and create heat). And, for reference's sake, I think 150V is too high a voltage. My lab recommends you run them at no higher than 120V (I run mine at 100V, but I'm cautious like that).

Hope that helps a bit!

#4 bob1

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Posted 08 October 2010 - 02:18 PM

The general rule for gels is 3-8 V/cm.

#5 Ameya P

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Posted 12 October 2010 - 03:04 AM

The general rule for gels is 3-8 V/cm.


About this, the length being measured is of the gel or is it the distance between the two electrodes?

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#6 donny

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Posted 12 October 2010 - 05:53 AM

It's the length between electrodes

#7 chromatin

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Posted 12 October 2010 - 05:21 PM

Prepare your TBE buffer as below.
http://www.bioprotocols.info/reagent_and_buffer_recipes/10X-TBE.php
EDTA comes as sodium salt. If you have to much salt gel will heat up.
Good luck.




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