White big cloud in agar gel electrophoresis - (Apr/16/2012 )

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.

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...

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?

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.

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?

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

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?

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...)

leelee on Wed Apr 18 05:11:24 2012 said:

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.

bob1 on Wed Apr 18 08:22:02 2012 said:

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.