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Agarose gel electrophoresis troubleshooting


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37 replies to this topic

#16 fysio lab

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Posted 29 July 2010 - 12:16 AM

Maybe stupid insinuation: i hope the UV-illuminator is still working? UV-bulbs age and can be the reason for seeing faint bands?

#17 liugang

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Posted 25 August 2010 - 07:08 AM

perhaps the EB is unavailable and out of date so please try a new one

#18 gyma

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Posted 29 August 2010 - 09:59 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...

if so it sounds like there is something wrong with your etbr. I have never done this before though, do you think its possible to mix your sample with a little amount of etbr and check it under the UV.

#19 DNAgeek

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Posted 03 September 2010 - 06:16 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.
http://www.bioprotocols.info


That's what I was about to suggest, try a different EtBr stock.
You can see the EtBr running in the opposite durection than the sample, so the bottom of the gel is much less bright. Are you running your samples long and out of the EtBr? 2% gel is pretty thick, I usually run 1%, otherwise the samples run slowly and the EtBr may run out of the gel by the time you're done. Good luck

#20 Adrian K

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Posted 15 September 2010 - 04:31 AM

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!!!


Do you mean after they prepare the TBE solution, they add EtBr inside directly, and you just add in your agarose powder, microwave and mold it?
I thought you should only add EtBr after microwave and before you mold it? This is how I do my gel.

Edited by adrian kohsf, 15 September 2010 - 04:33 AM.

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#21 sameerbau

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Posted 21 November 2010 - 10:33 AM

i'd suggest two things:

1.if u're putting EtBR in the microwave-melted agarose in TBE/TAE..don't put it until the solution is cool!if u put EtBr when its boiling hot..it will degrade.

2.Try changing the loading dye?maybe it has too less glycerol?

#22 Ivanov_br

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Posted 22 November 2010 - 03:28 PM

Hi there!

I'm working on a new lab and i'm having a problem with my gel.

Why my DNA samples are running like this (see the figure)? The two lanes are 100 bp DNA ladder. All samples look the same. 1% Agarose gel, 80V 90 mA. I just don't have enough resolution with small fragments.

Posted Image

Brand new DNA ladder (promega)
Brand new agarose
Buffer TAE

Any ideas? Suggestions?

Thanks in advance!!!

Edited by Ivanov_br, 22 November 2010 - 03:29 PM.


#23 phage434

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Posted 22 November 2010 - 06:15 PM

1% agarose will not resolve 100 bp fragments well under any circumstances. Try 2% agarose, or (better) 3% Nusieve 3:1 agarose.

#24 Adrian K

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Posted 22 November 2010 - 06:49 PM

Did you run your gel long enough?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#25 Ameya P

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Posted 22 November 2010 - 09:00 PM

I use a 2.5% agarose and use a 100bp ladder. We use Seakem Agarose here and for better resolution, I would suggest using metaphor. Anyways, just increase the % of agarose to 2 or 3 % and you should be fine. Also, you should either stain with EtBr longer or add more EtBr when you make the gel. With little resolution and enough EtBr, the bands are blinding bright but yours are giving a weak signal.

Ameya

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#26 Ivanov_br

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Posted 23 November 2010 - 07:55 AM

Thank you guys, I tried a new agarose, and the resuld was really better!

Thanks!

#27 ninana

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Posted 05 April 2011 - 08:57 AM

hi there,
I have a similar problem: I have nice bands of DNA and marker on top of the gel (the side where the wells are), but exactly at 1/2 of the gel, they disappear... So no bands there, at least the marker should be visible.
I use 0.5% TBE Buffer (pH about 7-8 according to pH paper), tried different loading dyes, and stain with texasred (tried 1/2 to 1.5 hours, with 10-20ul per 100ml buffer)...
Any suggestions why this happens?

thanks a lot

#28 nicoledwards9815

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Posted 14 April 2011 - 03:13 AM

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

VebXperts Bpo

#29 phage434

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Posted 14 April 2011 - 03:53 AM

Ninana, the problem you are having with half of the gel unstained is caused by migration of the dye towards the negative electrode, while the DNA is migrating toward the positive electrode. This leaves the lower part of the gel unstained, since there is little dye left in that region. You can solve this problem by adding dye to the buffer tank in the positive electrode region, or by post-staining the gel.

#30 ninana

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Posted 27 April 2011 - 06:48 AM

Hi,
thanks for the answer. But I do post stain the gel, so the dye is everywhere... but:

problem solved:

It was the agarose. At least when I opened a new agarose, I suddenly got better gels. Attached is a picture of the old agarose (left) with a 1kb and 50bp marker, and the new agarose (right), with 1kb marker, my PCR products and a 50bp marker. I run the 2 gels together, so same conditions.
Does anyone know why the old agarose didn't work anymore? Maybe it got contaminated?

Attached Thumbnails

  • 110426 WC_PCR Nr 10 labeled.jpg





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