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Agarose Gel Electrophoresis Problems


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#1 biochemist_78

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Posted 19 May 2010 - 04:10 AM

Hi everybody,
Lately I am having problems visualizing the DNA agarose gels(1-2%) under the UV illumination as well as on gel doc.
It seems like half of my gel is illuminated and half of it is dark. I prepare the agarose in 1X TAE buffer and add the EtBr to the molten agarose.
I make sure to run the gel long enough till the dye front reaches almost the end of the gel. I have also checked the pH of the 1X TAE which is ~7.5 and is prepared fresh and also the gel loading buffer is prepared fresh.

I know that there is way where we can stain the gel after running it by soaking it EtBr solution- I haven't tried that as I believe that to soak the agarose gel completely in the EtBr stain I would require huge amount of the stain at least around 15-20ml so I am not sure whether that's a good idea to do it and also what concentration of the EtBr should be used!!

If anybody can share their words of wisdom I would really appreciate it.

Thanks.

#2 mdfenko

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Posted 19 May 2010 - 06:28 AM

etbr migrates counter to the dna in electrophoresis (it is positively charged and dna is negatively charged). this is why you get the dark area.

you can either add etbr to the running buffer or post run stain (and destain) to solve your problem.
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#3 lab rat

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Posted 19 May 2010 - 06:32 AM

Hi biochemist,

yes, you will need a larger amount of liquid. The amount of EtBr depends on the concentration, but I used to put 5-10 ul into 20-30 ml and stain it with very gentle shaking for about an hour (and go to lunch). Destain about 1/2 hr and take the pic. I like SYBRsafe better.

To discard the liquid, check with your institution's chemical hygiene plan. You will probably need to collect it and filter out the EtBr prior to discard.

regards,

lab rat
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#4 ProteinWork

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Posted 19 May 2010 - 07:52 AM

It's because of EtBr running towards the cathode (black wired end) in electrophoresis. So overtime the EtBr in the bottom end of your gel will be depleted.

What I usually do is to add some EtBr in the anode (red wired end) chamber before electrophoresis. Just to make sure that you have enough EtBr throughout the gel even after the electrophoresis is done.

You don't need much EtBr, either. Usually in a 50ml gel solution I add about 3-3.5 ul of EtBr and right before electrophoresis (after sample loading), I add about 7ul of EtBr into the anode chamber (which I estimate to contain about 100ml of running buffer).




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