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White big cloud in agar gel electrophoresis

Troubleshooting

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#1 juanpabloq

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Posted 16 April 2012 - 03:26 PM

Hello to everyone. I've been having problem with electrophoresis the past 2 weeks. A white cloud appears in more than upper half of the gel as shown in the picture. I usually make 2% agarose gel with TAE 50x. I make a stock of 1500 mL andI use 50 ml of this solution for the gel, with 3ul of Etrb (10mg/mL). I load the wells with 6ul (5uL dna and 1 uL of dye) For the ladder I use 1 uL of dye and 2 uL of ladder. I dont know what the hell is going on. I decreased the load of Etrb to 3uL ( before I use 5uL), I bought a new dye but the problem persists, also tried changing exposure times in UV transilluminator. I read this could be due to RNA contamination? Because sometimes they use this table to get RNA also. Suggestions will be greatly appreaciated. Thank you very much.

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  • Attached File  gel2.pdf   585.83KB   283 downloads

Edited by juanpabloq, 16 April 2012 - 03:27 PM.


#2 bob1

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Posted 16 April 2012 - 05:23 PM

This is called ethidium shadow, and is caused by the ethidium bromide migrating in the opposite direction to the DNA (i.e. towards the -ve electrode). It can be fixed by either adding ethidium bromide to the running buffer or by post-staining your gel.

Note that EtBr is usually used at 0.5 ug/ml not the 20 ug/ml that you are using...

#3 juanpabloq

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Posted 16 April 2012 - 05:55 PM

Thank you for your answer Bob, someone in other lab told me the same. I dont understand how adding Etbr to the TAE would make a difference. I usually make the TAE, then the agarose gel, put it 1.30 min in the microwave (take it every 30 seconds and shake it) and after this put the Etbr. Also, I use a 3 ul of a 10 mg/mL EtBr solution do u think im using a bad amount?

#4 phage434

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Posted 17 April 2012 - 04:04 AM

Bob was saying to add the EtBr to the buffer in your gel apparatus, in addition to the buffer used to make the gel. You only need it at the positive electrode. The EtBr in the running buffer replaces the EtBr which is migrating in the gel.

#5 juanpabloq

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Posted 17 April 2012 - 08:18 AM

Thank you for the advise Phage, im trying this now, will post if i get results or not later, thank you, what about PCR, could inapropiate cycle times in PCR lead to this kind of image later?

#6 juanpabloq

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Posted 17 April 2012 - 12:13 PM

This is the image i got today after adding EtBr at the running buffer and after being yelled by my supervisor for thinking this may cause cancer to everyone in the lab even though I covered the glass with tape. I think ill run another PCR

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#7 phage434

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Posted 17 April 2012 - 12:28 PM

OK. Much better. You are overloading too much DNA on the gel, but that is easy to fix. Your PCR is likely not working. I would say the bands you see are likely primer-dimers, or possibly just primers. Tell us more about your PCR reaction -- template, primers, expected size, cycling conditions, enzymes, everything. What ladder are you using?

#8 leelee

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Posted 17 April 2012 - 09:11 PM

Minor thread hijack here:

Your supervisor thinks adding EtBr in the tank buffer will cause cancer?

Exactly where does he/she think all of the EtBr in the gels migrates to anyway? You could try (gently) pointing out that the tanks are all already contaminated with EtBr and should therefore ALWAYS be handled with appropriate gloves anyway.

Not to mention that the EtBr/cancer hysteria is an overreaction. I'm yet to find any literature on any single case of cancer attributed to EtBr (happy to be corrected...but am doubting there is anything...)

#9 bob1

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Posted 18 April 2012 - 12:22 AM

Not to mention that the EtBr/cancer hysteria is an overreaction. I'm yet to find any literature on any single case of cancer attributed to EtBr (happy to be corrected...but am doubting there is anything...)

Not only that, ethidium bromide (under the name Homidium bromide) is/was used as an anti-trypanosome treatment (usually prophylactic - meaning continuous dosing) for cattle for many years, with no reported increase in cancers, even in the calves conceived during treatment. Several studies also have found no mutagenic effects in rats and mice, but I would have to dig around to find them again.

#10 hobglobin

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Posted 18 April 2012 - 08:48 AM


Not to mention that the EtBr/cancer hysteria is an overreaction. I'm yet to find any literature on any single case of cancer attributed to EtBr (happy to be corrected...but am doubting there is anything...)

Not only that, ethidium bromide (under the name Homidium bromide) is/was used as an anti-trypanosome treatment (usually prophylactic - meaning continuous dosing) for cattle for many years, with no reported increase in cancers, even in the calves conceived during treatment. Several studies also have found no mutagenic effects in rats and mice, but I would have to dig around to find them again.

I agree on that, with the only limitation that mice, rats and cattle live a few months or years....but we live 60-90 years (depending on longevity) and might work 20 or 40 years with EtBr...i.e. compared to these animals we have a much longer time of exposure and time to develop cancer...and nobody knows about the risks of chronic exposures...
Therefore though not becoming hysterical about it, I still want to wear nitrile gloves, work under a fume hood and try to avoid contaminations in the lab.

Edited by hobglobin, 18 April 2012 - 08:49 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#11 leelee

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Posted 18 April 2012 - 05:41 PM

True that our exposure is over a longer term than that of cattle administered EtBr prophylactically, but I also think it would be safe to assume that if cattle have no higher incidence of cancer at the massive doses they received (comparative to our incidental exposures), that the risk to us is equally negligible.

(I didn't do this calculation, rather read it on the blog of someone discussing this very issue)
"A 50kg researcher would need to drink 50,000 liters of gel-staining solution to get even the non-toxic dose used in cattle."

Interestingly, some of the new dyes purported to be "safer" than EtBr actually have higher toxicities in mouse studies than EtBr.

No harm in continuing to take precautions though :)

#12 swanny

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Posted 18 April 2012 - 06:57 PM

The problem I have encountered is that, after I point out the massive doses given to cattle, someone will come back and say "Ah, but what do they know about long-term, very low level doses?" , which is crazy talk to my mind . High-level doses do not have an acute toxicity, nor a long-term cancer-risk factor, so the body can handle EtBr. How do they think that the body can't process ultra-low levels? I mean, what route of entry do these people envisage? The BP of ethidium is over 200C, so there won't be any in the steam that can be generated, thus no aerosol problem. You could drink it or inject it, I suppose, but that's what they did to the cattle, and as has already been pointed out, neither the cattle, nor their offspring, nor the herdsmen who lived with and on the cattle developed cancers. What we need is an epidemiological study of cancer rates across 30 to 40 years in molecular biologists vs scientists who do not use EtBr. Any volunteers?

Sometimes I think I work in a sheltered workshop for intellectually gifted people...
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#13 hobglobin

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Posted 19 April 2012 - 09:06 AM

Well my idea on this is: It's a lab and I work carefully with chemicals...
EtBr diffuses through usual Latex gloves in a few seconds, and can be absorbed through the skin and aerosols don't need 200°C, but these tiny droplets are in steam of boiling water (to avoid this you boil the agarose without EtBr, because you're careful)...if a hood is really necessary then is surely arguable, but often prescribed.

EtBr has effects on animals (LD50 mice: 110 mg/kg with hypodermic injection; rats: 1503 mg/kg oral), the liver processes it and the metabolites are mutagen (too or alone). Here's a extensive webpage on studies about EtBr.
And I wonder if anybody ever checked all the treated cattle for tumours, I don't think so...anyway the effects on other species are a hint only and no proof that it's more or less toxic, teratogen or mutagen to humans. So I'm careful and avoid unnecessary contact.
According to a paper quoted in wikipedia it also acts as topoisomerase I poison similar to some anticancer drugs. Anyway do I want to take an anticancer drug? No.

I'm not hysteric about it (I work with EtBr almost every day), but careful and sceptic in both directions.

Edited by hobglobin, 19 April 2012 - 09:48 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#14 pito

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Posted 19 April 2012 - 10:43 AM

Well my idea on this is: It's a lab and I work carefully with chemicals...
EtBr diffuses through usual Latex gloves in a few seconds, and can be absorbed through the skin and aerosols don't need 200°C, but these tiny droplets are in steam of boiling water (to avoid this you boil the agarose without EtBr, because you're careful)...if a hood is really necessary then is surely arguable, but often prescribed.

EtBr has effects on animals (LD50 mice: 110 mg/kg with hypodermic injection; rats: 1503 mg/kg oral), the liver processes it and the metabolites are mutagen (too or alone). Here's a extensive webpage on studies about EtBr.
And I wonder if anybody ever checked all the treated cattle for tumours, I don't think so...anyway the effects on other species are a hint only and no proof that it's more or less toxic, teratogen or mutagen to humans. So I'm careful and avoid unnecessary contact.
According to a paper quoted in wikipedia it also acts as topoisomerase I poison similar to some anticancer drugs. Anyway do I want to take an anticancer drug? No.

I'm not hysteric about it (I work with EtBr almost every day), but careful and sceptic in both directions.


You are right.

But what I "dislike" is that EthBr. "gets a lot of attention" and people freak out whenever something goes "wrong" with EthBr and at the same time they use other chemicals (often more dangerous or as dangerous as EthBr.) without any precautions. Or often they think that EthBr is soo bad that the alternatives must be 100% safe and (figure of speach), they could drink it and they drop their gaurd and start contamination the entire lab with this alternative.

The reaction of that supervisor is classical and shows how people dont think enough.. The gels themself contain EthBr etc.. as mentioned before.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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