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Agarose gel electrophoresis troubleshooting


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#1 kdw

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Posted 01 May 2009 - 11:04 AM

Hi all,
I was wondering if someone could help. The last 3 days or so, my gels have been looking abysmal. The dye almost fades from the gel as it runs, so when I finally image it the bands are so faint that I have to use long (2s) exposure and then tone down the image intensity. Here are some details.
I use a 2% gel for bands that range from 250-600bp. I just switched to a new bottle of agarose, same brand tho. I use TAE buffer that I re-made but still have the problem. I usually make the gel, add EtBr and pour it into a mold. I load 10uL of sample into the wells after adding some 6x loading dye.

The bands, as the gel runs, just seem to fade. The ladder does not image well either, which makes me believe it's not my samples. I have re-made the TAE buffer and am now trying another lab's TBE buffer to see if that will help.

Anyone had a similar issue??

#2 gfischer

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Posted 01 May 2009 - 11:09 AM

Try adding EtBr to the running buffer as well. If you just add it to the gel, it will sometimes run right out of the gel.
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#3 HomeBrew

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Posted 01 May 2009 - 01:43 PM

Instead of adding EtBr to the gel, soak the gel in an EtBr solution after the run, then destain in distilled water.

What tracking dye are you using? Most tracking dyes are pH sensitive, so your "fading bands" might indicate a pH problem, which might further indicate a problem with your gel buffer.

You're not accidentally making your molten agarose with water, are you?

#4 kdw

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Posted 01 May 2009 - 01:58 PM

No, I'm not using water... I just tried a TBE solution prepared down the hall. They put EtBr in their stock, so I used it to make the gel and to run it in ... and it was still so faint! At the end of running it (I only even ran it for like 30 min) I could barely see the dye in the gel. I used a 2 s exposure and could see only the faintest bands.

And regarding the dye, I used one dye to mix with the samples and another dye to make the ladder... and both were not good.

I didn't check the pH of the TBE buffer. When I made the new TAE yesterday I made sure it was around 7.5 .

I am going to try the other labs' agarose next. It seems it HAS to be the buffer that's the issue - but even with all the changes I've made I don't know what could be wrong! Frustrating.

I will be back in on Monday to make a new gel. I am going to use the other lab's TBE AND their Agarose. Thanks for all the suggestions guys!!!

#5 research_freak

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Posted 07 May 2009 - 09:22 AM

Always make sure you use fresh buffer everytime you run a gel (or at least when you're running important samples).
If the buffer is reused often, it is not of much good.

#6 Lazinase

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Posted 07 May 2009 - 10:08 PM

I'm just wondering if using SYBR Green can be an alternative method or not...you know, EtBr is quite toxic...

I used SYBR Green and nothing like that happens...

#7 mdfenko

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Posted 14 May 2009 - 10:56 AM

...you know, EtBr is quite toxic...

etbr may not be as toxic as you think, check the links in this post.
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#8 OA17

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Posted 20 May 2009 - 01:18 PM

Hi!

I don't really think that the problem is with the buffer itself, because if you have changed it and you still have the problem, it must be elsewhere. I don't think that the problem should be with EtBr either. I usually put it in the gel, not in the loading buffer.

What I recommend you is to clean the cuvette, the mold and the comb with water and soap very well before using it. You may think this is a stupid thing, but if somebody has used it to run RNA samples, for example, and it has not been cleaned, some rests of formamide, etc, may remain, and this will spoil your gel for DNA.

Of course, check the pH of the running buffer, check that the loading buffer is OK, and so on.

Good luck!

Oh! I was forgetting to tell you that I prefer EtBr rather than Syber Safe because Syber Safe loses its intensity much faster that EtBr (at least from my experience).

#9 ickyostes

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Posted 11 June 2009 - 11:30 PM

You're looking at some pretty small fragments, and EtBr migrates in the opposite direction from DNA during electrophoresis. Because of that, you can get uneven ladder staining and inconsistent staining of fragments. I recommend staining in an EtBr bath after the run, also because this gives you sharper resolution and is better for quantification.

If you're wondering if the buffer is getting spent during the run, check its pH after the run both in front of and behind the gel. That should only really be a problem if you're doing a long run, though. TAE is fine for me up to a few hours, but is exhausted faster than TBE so I'd try TBE for longer runs. I've had to do some fairly long runs before, and for these longer runs the dye will dissipate and fade.

I'd also check the quality of your DNA to make sure you aren't getting protein or nuclease contamination. Salt concentration can also be an issue, so you could try ethanol-precipitation to clean that up.

#10 HomeBrew

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Posted 12 June 2009 - 12:36 PM

Good points, ickyostes!

I've never added EtBr to the molten agarose for just those reasons -- I've always (three different labs and 20+ years now) stained/destained my gels after the run, for just the reasons you point out.

Checking the pH of the buffer in the two different chambers after the run is a good idea, too...

#11 gebirgsziege

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Posted 19 June 2009 - 04:55 AM

I've never added EtBr to the molten agarose for just those reasons -- I've always (three different labs and 20+ years now) stained/destained my gels after the run, for just the reasons you point out.



I am always adding EtBr directly to the gel and always get beautiful gels; never had any problems with visualising my products (long and short ones). Worst case was that the gel had run dark, but the DNA was still brightly shining at me.

Although late, but another suggestion for the problem: have you checked the power supply???? I have faced a similar problem once: The loading dye was slowly diffunding into the gel and was fading away....so I found out that our power supply was broken.
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#12 warsel

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Posted 22 June 2009 - 05:59 AM

same here.. never had problems with staining the gel right before pouring, even with 70kb size fragments.

how long do you run your gels?
it might work better if you don't run them for too long.

could it be the problem you are seeing is actually high background (ie by adding too much etbr or by having a gel that is not clean (ie bad brand, dust in the agarose, re-used a gel too often)?

i have made the experience that many people actually add too much etbr to their gels, increasing the background and lowering the s/n ratio.

#13 krystle

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Posted 23 October 2009 - 06:38 AM

same here.. never had problems with staining the gel right before pouring, even with 70kb size fragments.

how long do you run your gels?
it might work better if you don't run them for too long.

could it be the problem you are seeing is actually high background (ie by adding too much etbr or by having a gel that is not clean (ie bad brand, dust in the agarose, re-used a gel too often)?

i have made the experience that many people actually add too much etbr to their gels, increasing the background and lowering the s/n ratio.



Hi warsel, can i check to confirm that you stained your agarose gel before casting, and while running such large fragments, you don't have problem with the separation of the large bands? i'm asking this because I'm looking to buy some large dna ladder and its recommended that i do post gel staining as doing pre-staining might affect separation of the larger bands of the ladder. Also, what the % of gel you use and conditions you run your gel for? Thanks in advance.

#14 sdar

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Posted 27 April 2010 - 08:47 AM

just add a little etbr in the tank buffer and the bfr running the gel the bands would not fade

#15 chromatin

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Posted 28 July 2010 - 05:39 PM

Ethidium bromide is light sensitive. Make sure that stock of it was kept in dark. If it's exposed to light long time, you will get very week signal.
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