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EtBr in agarose gel and running buffer

Agarose Gel DNA Electrophoresis EtBr

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#1 Epigeneticist

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Posted 13 May 2013 - 05:19 PM

I know there are mixed opinions on how to stain an DNA agarose gel with EtBr but I am not asking for people's preference in this post but rather hoping they would answer my questions. After cooling my melted agarose I add EtBr to a final concentration of 0.5ug/ml. I also add EtBr to my running buffer to make it roughly a similar concentration as my gel (I only add EtBr to running buffer if it fresh). We re-use the running buffer for gels and replace it after a few weeks or months when the gel resolution and migration drops or buffer gets funky. . So...

If I'm not changing out the buffer with every new gel I run, will the EtBr degrade over time and not stain the gel effectively (i.e. cause the EtBr front in bottom part of gel due to difference between EtBr in gel and buffer)?

Do I need to add EtBr after a certain period of time? Just not sure how often I should be adding it to the buffer that is re-used. I do not want to end up with a concentrated bath of EtBr but I also want to make sure the EtBr is effective at staining my gel evenly.



Again, I understand some people disagree with this staining process and would rather just do post-stain or only add EtBr to the gel so I hope people will focus on providing useful input to my specific questions.

Thanks!

#2 bob1

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Posted 13 May 2013 - 05:31 PM

There would possibly be some photo-degradation of the EtBr with time, but this should be fairly minimal unless the time elapsed is substantial (i.e. months). EtBr will also migrate under an electric field towards the negative electrode (cathode), so technically you will deplete the Etbr in the tank at the anode (+ electrode). You can just add EtBr to the anode to overcome this problem. The rate at which it migrates and hence depletes will depend on how long you are running the tanks for and how much charge (voltage) you are putting over them, so there is no "you need to add this amount at X time points" sort of answer.

#3 Epigeneticist

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Posted 13 May 2013 - 05:38 PM

Thanks Bob1!

Couldn't I just rock the gel chamber every so often to disperse the EtBr? The whole number of days thing was based on that fact I do not know the "photo-stability" of EtBr. Didn't know if the degradation rate was common knowledge that I was lacking. I just know that EtBr is always in a brown bottle and people keep post-stain in a light protected container so I figured it was sensitive to photo degradation.

#4 phage434

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Posted 14 May 2013 - 07:57 AM

Even if it degrades, the amount of EtBr needed for staining is miniscule -- much much less than you are adding.





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